Triple combination antibody therapies

ABSTRACT

The present invention is directed to triple combination therapies with anti-TIGIT antibodies, anti-PVRIG antibodies, and checkpoint inhibitors, including anti-PD-1 or anti-PD-L1 antibodies.

CROSS REFERENCE TO RELATED APPLICAITONS

This application is a continuation of U.S. patent application Ser. No.15/996,369 filed Jun. 1, 2018 which claims priority under 35 U.S.C. §119 to U.S. Patent Application Nos. 62/513,960 filed Jun. 1, 2017,62/515,452 filed Jun. 5, 2017, 62/538,563 filed Jul. 28, 2017,62/547,051 filed Aug. 17, 2017, 62/582,756 filed Nov. 7, 2017 and62/618,005 filed Jan. 16, 2018, all of which are expressly incorporatedherein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 9, 2022, isnamed 1143865009US01_SeqList and is 1442 KB in size.

I. BACKGROUND OF THE INVENTION

TIGIT is a coinhibitory receptor that is highly expressed on effector &regulatory (Treg) CD4+ T cells, effector CD8+ T cells, and NK cells.TIGIT has been shown to attenuate immune response by (1) directsignaling, (2) inducing ligand signaling, and (3) competition with anddisruption of signaling by the costimulatory receptor CD226 (also knownas DNAM-1). TIGIT signaling has been the most well-studied in NK cells,where it has been demonstrated that engagement with its cognate ligand,poliovirus receptor (PVR, also known as CD155) directly suppresses NKcell cytotoxicity through its cytoplasmic ITIM domain. Knockout of theTIGIT gene or antibody blockade of the TIGIT/PVR interaction has shownto enhance NK cell killing in vitro, as well as to exacerbate autoimmunediseases in vivo. In addition to its direct effects on T- and NK cells,TIGIT can induce PVR-mediated signaling in dendritic or tumor cells,leading to the increase in production of anti-inflammatory cytokinessuch as IL10. In T-cells TIGIT can also inhibit lymphocyte responses bydisrupting homodimerization of the costimulatory receptor CD226, and bycompeting with it for binding to PVR.

TIGIT is highly expressed on lymphocytes, including Tumor InfiltratingLymphocytes (TILs) and Tregs, that infiltrate different types of tumors.PVR is also broadly expressed in tumors, suggesting that the TIGIT-PVRsignaling axis may be a dominant immune escape mechanism for cancer.Notably, TIGIT expression is tightly correlated with the expression ofanother important coinhibitory receptor, PD1. TIGIT and PD1 areco-expressed on the TILs of numerous human and murine tumors. UnlikeTIGIT and CTLA4, PD1 inhibition of T cell responses does not involvecompetition for ligand binding with a costimulatory receptor.

The immune checkpoint, poliovirus receptor related immunoglobulin domaincontaining (PVRIG, also known as CD112R) represents a new inhibitoryreceptor within the TIGIT family of receptors. PVRIG binds with highaffinity to its cognate ligand, poliovirus receptor-related 2 (PVRL2,also known as CD112 or nectin-2) to deliver an inhibitory signal throughits ITIM motif within T and NK cells. The affinity of TIGIT to PVR andof PVRIG to PVRL2 is higher than the affinity of CD226 to either PVR orPVRL2, suggesting TIGIT and PVRIG can outcompete PVR and PVRL2 fromCD226 and providing an indirect mechanism by which TIGIT and PVRIG canreduce lymphocyte function. Thus, two receptors with the same family,TIGIT and PVRIG, deliver inhibitory signals to dampen T and NK cellresponses.

Accordingly, TIGIT and PVRIG are attractive for triple therapycombinations with checkpoint inhibitors, including anti-PD-1 antibodies.

II. BRIEF SUMMARY OF THE INVENTION

The present invention provides methods and compositions comprisingcombinations of the TIGIT antibodies as disclosed herein and as providedin the claims with PVRIG antibodies and checkpoint inhibitors, includinganti-PD-1 antibodies. The present invention also provides for nucleicacids encoding said antibodies and compositions thereof.

The present invention provides a method of treating cancer said patientcomprising: a) providing a biopsy from said patient comprising tumorcells; b) measuring the frequency of PD-L1 positive tumor cells orimmune cells in said biopsy; c) if said frequency of PD-L1 positivetumor cells or immune cells is greater than 1% compared to staining thesame tumor cells with a relevant isotype control antibody for theantibodies used, administering a triple combination therapy comprisingan anti-TIGIT antibody, an anti-PVRIG antibody and an anti-PD-1antibody; and d) if said frequency of PD-L1 positive tumor cells orimmune cells is less than 1% compared to staining the same tumor cellswith a relevant isotype control antibody for the antibodies used,administering a double combination therapy comprising an anti-TIGITantibody and an anti-PVRIG antibody.

In some embodiments of the method, anti-TIGIT antibody is an antibodychosen from at least one of CPA.9.083.H4(S241P), CPA.9.086.H4(S241P),CHA.9.547.7.H4(S241P) and CHA.9.547.13.H4(S241P).

In some embodiments of the method, anti-PVRIG antibody is an antibodychosen from at least one of CHA.7.518.1.H4(S241P) andCHA.7.538.1.2.H4(S241P).

In some embodiments of the method, the anti-PD-1 antibody is an antibodychosen from at least one of pembrolizumab, cemiplimab, and nivolumab.

In some embodiments of the method, the double combination therapy ischosen from the administration of CPA.9.083.H4(S241P) andCHA.7.518.1.H4(S241P); CPA.9.086.H4(S241P) and CHA.7.518.1.H4(S241P);CHA.9.547.7.H4(S241P) and CHA.7.518.1.H4(S241P); CHA.9.547.13.H4(S241P)and CHA.7.518.1.H4(S241P); CPA.9.083.H4(S241P) andCHA.7.538.1.2.H4(S241P); CPA.9.086.H4(S241P) andCHA.7.538.1.2.H4(S241P); CHA.9.547.7.H4(S241P) andCHA.7.538.1.2.H4(S241P); and CHA.9.547.13.H4(S241P) andCHA.7.538.1.2.H4(S241P).

In some embodiments of the method, the triple combination therapy ischosen from the administration of CPA.9.083.H4(S241P), pembrolizumab andCHA.7.518.1.H4(S241P); CPA.9.086.H4(S241P), pembrolizumab andCHA.7.518.1.H4(S241P); CHA.9.547.7.H4(S241P), pembrolizumab andCHA.7.518.1.H4(S241P); CHA.9.547.13.H4(S241P), pembrolizumab andCHA.7.518.1.H4(S241P); CPA.9.083.H4(S241P), pembrolizumab andCHA.7.538.1.2.H4(S241P); CPA.9.086.H4(S241P), pembrolizumab andCHA.7.538.1.2.H4(S241P); CHA.9.547.7.H4(S241P), pembrolizumab andCHA.7.538.1.2.H4(S241P); CHA.9.547.13.H4(S241P), pembrolizumab andCHA.7.538.1.2.H4(S241P); CPA.9.083.H4(S241P), nivolumab andCHA.7.518.1.H4(S241P); CPA.9.086.H4(S241P), nivolumab andCHA.7.518.1.H4(S241P); CHA.9.547.7.H4(S241P), nivolumab andCHA.7.518.1.H4(S241P); CHA.9.547.13.H4(S241P), nivolumab andCHA.7.518.1.H4(S241P); CPA.9.083.H4(S241P), nivolumab andCHA.7.538.1.2.H4(S241P); CPA.9.086.H4(S241P), nivolumab andCHA.7.538.1.2.H4(S241P); CHA.9.547.7.H4(S241P, nivolumab andCHA.7.538.1.2.H4(S241P); CHA.9.547.13.H4(S241P), nivolumab andCHA.7.538.1.2.H4(S241P); CHA.7.538.1.2.H4(S241P); CPA.9.083.H4(S241P),cemiplimab and CHA.7.518.1.H4(S241P); CPA.9.086.H4(S241P), cemiplimaband CHA.7.518.1.H4(S241P); CHA.9.547.7.H4(S241P), cemiplimab andCHA.7.518.1.H4(S241P); CHA.9.547.13.H4(S241P), cemiplimab andCHA.7.518.1.H4(S241P); CPA.9.083.H4(S241P), cemiplimab andCHA.7.538.1.2.H4(S241P); CPA.9.086.H4(S241P), cemiplimab andCHA.7.538.1.2.H4(S241P); CHA.9.547.7.H4(S241P, cemiplimab andCHA.7.538.1.2.H4(S241P); and CHA.9.547.13.H4(S241P), cemiplimab andCHA.7.538.1.2.H4(S241P).

In some embodiments of the method, the antibodies are administeredsimultaneously.

In some embodiments of the method, the antibodies are administeredsequentially.

In some embodiments of the method, the cancer is selected from the groupconsisting of prostate cancer, liver cancer (HCC), colorectal cancer,ovarian cancer, endometrial cancer, breast cancer, triple negativebreast cancer, pancreatic cancer, stomach (gastric) cancer, cervicalcancer, head and neck cancer, thyroid cancer, testis cancer, urothelialcancer, lung cancer (small cell lung, non-small cell lung), melanoma,non melanoma skin cancer (squamous and basal cell carcinoma), glioma,renal cancer (RCC), lymphoma (NHL or HL), Acute myeloid leukemia (AML),T cell Acute Lymphoblastic Leukemia (T-ALL), Diffuse Large B celllymphoma, testicular germ cell tumors, mesothelioma, esophageal cancer,Merkel Cells cancer, MSI-high cancer, KRAS mutant tumors, adult T-cellleukemia/lymphoma, and Myelodysplastic syndromes (MDS). In someembodiments of the method, the cancer is selected from the groupconsisting of cancer triple negative breast cancer, stomach (gastric)cancer, lung cancer (small cell lung, non-small cell lung), Merkel Cellscancer, MSI-high cancer, KRAS mutant tumors, adult T-cellleukemia/lymphoma, and Myelodysplastic syndromes (MDS). In someembodiments of the method, the cancer is selected from the groupconsisting of cancer triple negative breast cancer, stomach (gastric)cancer, lung cancer (small cell lung, non-small cell lung), Merkel Cellscancer, and MSI-high cancer.

The present invention also provides a method of treating cancer saidpatient comprising administering a triple combination therapy comprisingan anti-TIGIT antibody, an anti-PVRIG antibody, and an anti-PD-1antibody.

In some embodiments of the method, the anti-TIGIT antibody is anantibody chosen from at least one of CPA.9.083.H4(S241P),CPA.9.086.H4(S241P), CHA.9.547.7.H4(S241P), and CHA.9.547.13.H4(S241P).

In some embodiments of the method, the anti-PVRIG antibody is anantibody chosen from at least one of CHA.7.518.1.H4(S241P) andCHA.7.538.1.2.H4(S241P).

In some embodiments of the method, the anti-PD-1 antibody is an antibodychosen from at least one of pembrolizumab, cemiplimab and nivolumab.

In some embodiments of the method, the triple combination therapycomprises the administration of an anti-PD-1 antibody in combinationwith a double-combination therapy chosen from the administration ofCPA.9.083.H4(S241P) and CHA.7.518.1.H4(S241P); CPA.9.086.H4(S241P) andCHA.7.518.1.H4(S241P); CHA.9.547.7.H4(S241P) and CHA.7.518.1.H4(S241P);CHA.9.547.13.H4(S241P) and CHA.7.518.1.H4(S241P); CPA.9.083.H4(S241P)and CHA.7.538.1.2.H4(S241P); CPA.9.086.H4(S241P) andCHA.7.538.1.2.H4(S241P); CHA.9.547.7.H4(S241P) andCHA.7.538.1.2.H4(S241P); and CHA.9.547.13.H4(S241P) andCHA.7.538.1.2.H4(S241P).

In some embodiments of the method, the triple combination therapy ischosen from the administration of CPA.9.083.H4(S241P), pembrolizumab andCHA.7.518.1.H4(S241P); CPA.9.086.H4(S241P), pembrolizumab andCHA.7.518.1.H4(S241P); CHA.9.547.7.H4(S241P), pembrolizumab andCHA.7.518.1.H4(S241P); CHA.9.547.13.H4(S241P), pembrolizumab andCHA.7.518.1.H4(S241P); CPA.9.083.H4(S241P), pembrolizumab andCHA.7.538.1.2.H4(S241P); CPA.9.086.H4(S241P), pembrolizumab andCHA.7.538.1.2.H4(S241P); CHA.9.547.7.H4(S241P), pembrolizumab andCHA.7.538.1.2.H4(S241P); CHA.9.547.13.H4(S241P), pembrolizumab andCHA.7.538.1.2.H4(S241P); CPA.9.083.H4(S241P), nivolumab andCHA.7.518.1.H4(S241P); CPA.9.086.H4(S241P), nivolumab andCHA.7.518.1.H4(S241P); CHA.9.547.7.H4(S241P), nivolumab andCHA.7.518.1.H4(S241P); CHA.9.547.13.H4(S241P), nivolumab andCHA.7.518.1.H4(S241P); CPA.9.083.H4(S241P), nivolumab andCHA.7.538.1.2.H4(S241P); CPA.9.086.H4(S241P), nivolumab andCHA.7.538.1.2.H4(S241P); CHA.9.547.7.H4(S241P, nivolumab andCHA.7.538.1.2.H4(S241P); CHA.9.547.13.H4(S241P), nivolumab andCHA.7.538.1.2.H4(S241P); CHA.7.538.1.2.H4(S241P); CPA.9.083.H4(S241P),cemiplimab and CHA.7.518.1.H4(S241P); CPA.9.086.H4(S241P), cemiplimaband CHA.7.518.1.H4(S241P); CHA.9.547.7.H4(S241P), cemiplimab andCHA.7.518.1.H4(S241P); CHA.9.547.13.H4(S241P), cemiplimab andCHA.7.518.1.H4(S241P); CPA.9.083.H4(S241P), cemiplimab andCHA.7.538.1.2.H4(S241P); CPA.9.086.H4(S241P), cemiplimab andCHA.7.538.1.2.H4(S241P); CHA.9.547.7.H4(S241P, cemiplimab andCHA.7.538.1.2.H4(S241P); and CHA.9.547.13.H4(S241P), cemiplimab andCHA.7.538.1.2.H4(S241P).

In some embodiments of the method, the antibodies are administeredsimultaneously.

In some embodiments of the method, the antibodies are administeredsequentially.

In some embodiments of the method, the cancer for the triple combinationtherapy is selected from the group consisting of prostate cancer, livercancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer,breast cancer, triple negative breast cancer, pancreatic cancer, stomach(gastric) cancer, cervical cancer, head and neck cancer, thyroid cancer,testis cancer, urothelial cancer, lung cancer (small cell lung,non-small cell lung), melanoma, non melanoma skin cancer (squamous andbasal cell carcinoma), glioma, renal cancer (RCC), lymphoma (NHL or HL),Acute myeloid leukemia (AML), T cell Acute Lymphoblastic Leukemia(T-ALL), Diffuse Large B cell lymphoma, testicular germ cell tumors,mesothelioma, esophageal cancer, Merkel Cells cancer, MSI-high cancer,KRAS mutant tumors, adult T-cell leukemia/lymphoma, and Myelodysplasticsyndromes (MDS). In some embodiments of the method, the cancer isselected from the group consisting of cancer triple negative breastcancer, stomach (gastric) cancer, lung cancer (small cell lung,non-small cell lung), Merkel Cells cancer, MSI-high cancer, KRAS mutanttumors, adult T-cell leukemia/lymphoma, and Myelodysplastic syndromes(MDS).

The present invention also provides a pharmaceutical dose kitcomprising: a) a container comprising a unit dosage of an anti-TIGITantibody; and b) a container comprising a unit dosage of an anti- PVRIGantibody.

The present invention also provides a pharmaceutical dose kitcomprising: a) a container comprising a unit dosage of an anti-TIGITantibody; b) a container comprising a unit dosage of an anti- PVRIGantibody; and c) a container comprising an anti-PD-1 antibody.

In a further aspect, the invention provides methods comprising: a)providing a cell population from a tumor sample from a patient; b)staining said population with labeled antibodies that bind: i) TIGITprotein; ii) PVRIG protein; iii) PVR protein; iv) PD-1 protein; v) PD-L1protein; vi) PVRL2; and vi) a relevant isotype control for theantibodies in i)-vi); c) running fluorescence activated cell sorting(FACS); d) for each of TIGIT, PVRIG, PVR, PD-1, PVRL2 and PD-L1,determining the percentage of cells in said population that express theprotein relative to said isotype control antibody; wherein if thepercentage of positive cells is >1% for either TIGIT or PVR, and foreither PVRIG or PVRL2, and for either PD-1 or PD-L1, proceeding to stepe); and e) administering antibodies to TIGIT, PVRIG, and PD-1 to saidpatient.

In a further aspect, the invention provides methods comprising: a)providing a cell population from a tumor sample from a patient; b)staining said population with labeled antibodies that bind: i) TIGITprotein; ii) PVR protein; iii) PD-1 protein; iv) PD-L1 protein; and v) arelevant isotype control for the antibodies in i)-iv); c) runningfluorescence activated cell sorting (FACS); d) for each of TIGIT, PVR,PD-1, and PD-L1, determining the percentage of cells in said populationthat express the protein relative to said isotype control antibody;wherein if the percentage of positive cells is >1% for all 4 receptors,e) administering antibodies to TIGIT and PD-1 to said patient.

In an additional aspect, the invention provides methods comprising: a)providing a cell population from a tumor sample from a patient; b)staining said population with labeled antibodies that bind: i) PVRIGprotein; ii) PVRL2 protein; iii) PD-1 protein; iv) PD-L1 protein; and v)a relevant isotype control for the antibodies in i)-iv); c) runningfluorescence activated cell sorting (FACS); d) for each of PVRIG, PVRL2,PD-1 and PD-L1, determining the percentage of cells in said populationthat express the protein relative to said isotype control antibody;wherein if the percentage of positive cells is >1% for all 4 receptors,e) administering antibodies to PVRIG and PD-1 to said patient.

In a further aspect, the invention provides methods comprising a)providing a cell population from a tumor sample from a patient; b)staining said population with labeled antibodies that bind: i) PVRIGprotein; ii) PVRL2 protein; iii) TIGIT protein; iv) PVR protein; and v)a relevant isotype control for the antibodies in i)-iv); c) runningfluorescence activated cell sorting (FACS); d) for each of PVRIG, PVRL2,TIGIT and PVR, determining the percentage of cells in said populationthat express the protein relative to said isotype control antibody;wherein if the percentage of positive cells is >1% for all 4 receptors,e) administering antibodies to PVRIG and TIGIT to said patient.

In an additional aspect, the invention provides methods comprising: a)providing a cell population from a tumor sample from a patient; b)staining said population with labeled antibodies that bind: i) PVRIGprotein; ii) TIGIT protein; iii) PVRL2 protein; iv) PD-1 protein; v)PD-L1 protein; and vi) a relevant isotype control for the antibodies ini)-v); c) running fluorescence activated cell sorting (FACS); d) foreach of PVRIG, TIGIT, PVRL2, PD-1 and PD-L1, determining the percentageof cells in said population that express the protein relative to saidisotype control antibody; wherein if the percentage of positive cellsis >1% for all 5 receptors, e) administering antibodies to PVRIG, TIGIT,and PD-1 to said patient.

In a further aspect, the invention provides methods comprising a)providing a cell population from a tumor sample from a patient; b)staining said population with labeled antibodies that bind: i) PVRIGprotein; ii) PVRL2 protein; iii) TIGIT protein; iv) PVR protein; v)PD-1; and vi) a relevant isotype control for the antibodies in i)-v); c)running fluorescence activated cell sorting (FACS); d) for each ofPVRIG, PVRL2, TIGIT and PVR, determining the percentage of cells in saidpopulation that express the protein relative to said isotype controlantibody; wherein if the percentage of positive cells is >1% for all 4receptors, e) administering antibodies to PVRIG, TIGIT, and PD-1 to saidpatient.

In some embodiments, the present invention provides a method of treatingcancer in a patient comprising: a) providing a biopsy from said patientcomprising tumor cells; b) measuring the frequency of PD-L1 positivetumor cells or immune cells in said biopsy; c) if said frequency ofPD-L1 positive tumor cells or immune cells is greater than 1% comparedto staining the same tumor cells with a relevant isotype controlantibody for the antibodies used, administering a triple combinationtherapy comprising an anti-TIGIT antibody, an anti-PVRIG antibody and ananti-PD-1 antibody; and d) if said frequency of PD-L1 positive tumorcells or immune cells is less than 1% compared to staining the sametumor cells with a relevant isotype control antibody for the antibodiesused, administering a double combination therapy comprising ananti-TIGIT antibody and an anti-PVRIG antibody.

In some embodiments, the anti-TIGIT antibody is an antibody chosen fromany anti-TIGIT antibody described herein, including any of thosedescribed in FIG. 3 .

3 In some embodiments, the anti-PVRIG antibody is an antibody chosenfrom any anti-PVRIG antibody described herein, including any of thosedescribed in FIG. 5 and/or FIG. 63 .

In some embodiments, the anti-PD-1 antibody is an antibody chosen fromany anti-PD-1 antibody described herein, including any of thosedescribed in FIG. 7 .

In some embodiments, the anti-TIGIT antibody is an antibody chosen fromat least one of CPA.9.083.H4(S241P), CPA.9.086.H4(S241P),CHA.9.547.7.H4(S241P), and CHA.9.547.13.H4(S241P).

In some embodiments, the anti-PVRIG antibody is an antibody chosen fromat least one of CHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P).

In some embodiments, the anti-PD-1 antibody is an antibody chosen fromat least one of pembrolizumab, Cemiplimab and nivolumab.

8 In some embodiments, the double combination therapy is chosen from theadministration of CPA.9.083.H4(S241P) and CHA.7.518.1.H4(S241P);CPA.9.086.H4(S241P) and CHA.7.518.1.H4(S241P); CHA.9.547.7.H4(S241P) andCHA.7.518.1.H4(S241P); CHA.9.547.13.H4(S241P) and CHA.7.518.1.H4(S241P);CPA.9.083.H4(S241P) and CHA.7.538.1.2.H4(S241P); CPA.9.086.H4(S241P) andCHA.7.538.1.2.H4(S241P); CHA.9.547.7.H4(S241P) andCHA.7.538.1.2.H4(S241P); and CHA.9.547.13.H4(S241P) andCHA.7.538.1.2.H4(S241P).

In some embodiments, the triple combination therapy is chosen from theadministration of CPA.9.083.H4(S241P), pembrolizumab andCHA.7.518.1.H4(S241P); CPA.9.086.H4(S241P), pembrolizumab andCHA.7.518.1.H4(S241P); CHA.9.547.7.H4(S241P), pembrolizumab andCHA.7.518.1.H4(S241P); CHA.9.547.13.H4(S241P), pembrolizumab andCHA.7.518.1.H4(S241P); CPA.9.083.H4(S241P), pembrolizumab andCHA.7.538.1.2.H4(S241P); CPA.9.086.H4(S241P), pembrolizumab andCHA.7.538.1.2.H4(S241P); CHA.9.547.7.H4(S241P), pembrolizumab andCHA.7.538.1.2.H4(S241P); CHA.9.547.13.H4(S241P), pembrolizumab andCHA.7.538.1.2.H4(S241P); CPA.9.083.H4(S241P), nivolumab andCHA.7.518.1.H4(S241P); CPA.9.086.H4(S241P), nivolumab andCHA.7.518.1.H4(S241P); CHA.9.547.7.H4(S241P), nivolumab andCHA.7.518.1.H4(S241P); CHA.9.547.13.H4(S241P), nivolumab andCHA.7.518.1.H4(S241P); CPA.9.083.H4(S241P), nivolumab andCHA.7.538.1.2.H4(S241P); CPA.9.086.H4(S241P), nivolumab andCHA.7.538.1.2.H4(S241P); CHA.9.547.7.H4(S241P, nivolumab andCHA.7.538.1.2.H4(S241P); CHA.9.547.13.H4(S241P), nivolumab andCHA.7.538.1.2.H4(S241P); CHA.7.538.1.2.H4(S241P); CPA.9.083.H4(S241P),cemiplimab and CHA.7.518.1.H4(S241P); CPA.9.086.H4(S241P), cemiplimaband CHA.7.518.1.H4(S241P); CHA.9.547.7.H4(S241P), cemiplimab andCHA.7.518.1.H4(S241P); CHA.9.547.13.H4(S241P), andCHA.9.547.13.H4(S241P), and CHA.7.518.1.H4(S241P); CPA.9.083.H4(S241P),cemiplimab and CHA.7.538.1.2.H4(S241P); CPA.9.086.H4(S241P), cemiplimaband CHA.7.538.1.2.H4(S241P); CHA.9.547.7.H4(S241P, cemiplimab andCHA.7.538.1.2.H4(S241P); and CHA.9.547.13.H4(S241P), cemiplimab andCHA.7.538.1.2.H4(S241P).

In some embodiments, the antibodies are administered simultaneously.

In some embodiments, the antibodies are administered sequentially.

In some embodiments, the cancer is selected from the group consisting ofprostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer,endometrial cancer, breast cancer, triple negative breast cancer,pancreatic cancer, stomach (gastric) cancer, cervical cancer, head andneck cancer, thyroid cancer, testis cancer, urothelial cancer, lungcancer (small cell lung, non-small cell lung), melanoma, non melanomaskin cancer (squamous and basal cell carcinoma), glioma, renal cancer(RCC), lymphoma (NHL or HL), Acute myeloid leukemia (AML), T cell AcuteLymphoblastic Leukemia (T-ALL), Diffuse Large B cell lymphoma,testicular germ cell tumors, mesothelioma, esophageal cancer, MerkelCells cancer, MSI-high cancer, KRAS mutant tumors, adult T-cellleukemia/lymphoma, and Myelodysplastic syndromes (MDS).

In some embodiments, the cancer is selected from the group consisting oftriple negative breast cancer, stomach (gastric) cancer, lung cancer(small cell lung, non-small cell lung), and Merkel Cells cancer,MSI-high cancer KRAS mutant tumors, adult T-cell leukemia/lymphoma, andMyelodysplastic syndromes (MDS).

In some embodiments, the present invention provides a method of treatingcancer in a patient comprising administering a triple combinationtherapy comprising an anti-TIGIT antibody, an anti-PVRIG antibody, andan anti-PD-1 antibody.

In some embodiments, the anti-TIGIT antibody is an antibody chosen fromat least oneof CPA.9.083.H4(S241P), CPA.9.086.H4(S241P),CHA.9.547.7.H4(S241P), and CHA.9.547.13.H4(S241P).

In some embodiments, the said anti-PVRIG antibody is an antibody chosenfrom at least one of CHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P).

1 In some embodiments, the anti-PD-1 antibody is an antibody selectedfrom the group consisting of pembrolizumab, cemiplimab, and nivolumab.

In some embodiments, the triple combination therapy comprises theadministration of an anti-PD-1 antibody in combination with adouble-combination therapy chosen from the administration ofCPA.9.083.H4(S241P) and CHA.7.518.1.H4(S241P); CPA.9.086.H4(S241P) andCHA.7.518.1.H4(S241P); CHA.9.547.7.H4(S241P) and CHA.7.518.1.H4(S241P);CHA.9.547.13.H4(S241P) and CHA.7.518.1.H4(S241P); CPA.9.083.H4(S241P)and CHA.7.538.1.2.H4(S241P); CPA.9.086.H4(S241P) andCHA.7.538.1.2.H4(S241P); CHA.9.547.7.H4(S241P) andCHA.7.538.1.2.H4(S241P); and CHA.9.547.13.H4(S241P) andCHA.7.538.1.2.H4(S241P).

In some embodiments, the triple combination therapy is chosen from theadministration of CPA.9.083.H4(S241P), pembrolizumab andCHA.7.518.1.H4(S241P); CPA.9.086.H4(S241P), pembrolizumab andCHA.7.518.1.H4(S241P); CHA.9.547.7.H4(S241P), pembrolizumab andCHA.7.518.1.H4(S241P); CHA.9.547.13.H4(S241P), pembrolizumab andCHA.7.518.1.H4(S241P); CPA.9.083.H4(S241P), pembrolizumab andCHA.7.538.1.2.H4(S241P); CPA.9.086.H4(S241P), pembrolizumab andCHA.7.538.1.2.H4(S241P); CHA.9.547.7.H4(S241P), pembrolizumab andCHA.7.538.1.2.H4(S241P); CHA.9.547.13.H4(S241P), pembrolizumab andCHA.7.538.1.2.H4(S241P); CPA.9.083.H4(S241P), nivolumab andCHA.7.518.1.H4(S241P); CPA.9.086.H4(S241P), nivolumab andCHA.7.518.1.H4(S241P); CHA.9.547.7.H4(S241P), nivolumab andCHA.7.518.1.H4(S241P); CHA.9.547.13.H4(S241P), nivolumab andCHA.7.518.1.H4(S241P); CPA.9.083.H4(S241P), nivolumab andCHA.7.538.1.2.H4(S241P); CPA.9.086.H4(S241P), nivolumab andCHA.7.538.1.2.H4(S241P); CHA.9.547.7.H4(S241P, nivolumab andCHA.7.538.1.2.H4(S241P); CHA.9.547.13.H4(S241P), nivolumab andCHA.7.538.1.2.H4(S241P); CHA.7.538.1.2.H4(S241P); CPA.9.083.H4(S241P),cemiplimab and CHA.7.518.1.H4(S241P); CPA.9.086.H4(S241P), cemiplimaband CHA.7.518.1.H4(S241P); CHA.9.547.7.H4(S241P), cemiplimab andCHA.7.518.1.H4(S241P); CHA.9.547.13.H4(S241P), cemiplimab andCHA.7.518.1.H4(S241P); CPA.9.083.H4(S241P), cemiplimab andCHA.7.538.1.2.H4(S241P); CPA.9.086.H4(S241P), cemiplimab andCHA.7.538.1.2.H4(S241P); CHA.9.547.7.H4(S241P, cemiplimab andCHA.7.538.1.2.H4(S241P); and CHA.9.547.13.H4(S241P), cemiplimab andCHA.7.538.1.2.H4(S241P).

In some embodiments, the antibodies are administered simultaneously.

In some embodiments, the antibodies are administered sequentially.

In some embodiments, the cancer is selected from the group consisting ofprostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer,endometrial cancer, breast cancer, triple negative breast cancer,pancreatic cancer, stomach (gastric) cancer, cervical cancer, head andneck cancer, thyroid cancer, testis cancer, urothelial cancer, lungcancer (small cell lung, non-small cell lung), melanoma, non melanomaskin cancer (squamous and basal cell carcinoma), glioma, renal cancer(RCC), lymphoma (NHL or HL), Acute myeloid leukemia (AML), T cell AcuteLymphoblastic Leukemia (T-ALL), Diffuse Large B cell lymphoma,testicular germ cell tumors, mesothelioma, esophageal cancer, MerkelCells cancer, MSI-high cancer, KRAS mutant tumors, adult T-cellleukemia/lymphoma, and Myelodysplastic syndromes (MDS).

In some embodiments, the cancer is selected from the group consisting oftriple negative breast cancer, stomach (gastric) cancer, lung cancer(small cell lung, non-small cell lung), and Merkel Cells cancer,MSI-high cancer, KRAS mutant tumors, adult T-cell leukemia/lymphoma, andMyelodysplastic syndromes (MDS).

In some embodiments, the present invention provides a pharmaceuticaldose kit comprising: a) a container comprising a unit dosage of ananti-TIGIT antibody; and b) a container comprising a unit dosage of ananti-PVRIG antibody.

In some embodiments, the present invention provides a pharmaceuticaldose kit comprising: a) a container comprising a unit dosage of ananti-TIGIT antibody; b) a container comprising a unit dosage of ananti-PVRIG antibody; and c) a container comprising an anti-PD-1antibody.

In some embodiments, the anti-TIGIT antibody is an antibody chosen fromany anti-TIGIT antibody described herein, including any of thosedescribed in FIG. 3 .

In some embodiments, the anti-PVRIG antibody is an antibody chosen fromany anti-PVRIG antibody described herein, including any of thosedescribed in FIG. 5 and/or FIG. 63 .

In some embodiments, the anti-PD-1 antibody is an antibody chosen fromany anti-PD-1 antibody described herein, including any of thosedescribed in FIG. 7 .

In some embodiments, the present invention provides a method comprising:a) providing a cell population from a tumor sample from a patient; b)staining said population with labeled antibodies that bind: i) TIGITprotein; ii) PVRIG protein; iii) PVR protein; iv) PD-1 protein; v) PD-L1protein; and vi) a relevant isotype control for the antibodies in i)-v);c) running fluorescence activated cell sorting (FACS); d) for each ofTIGIT, PVRIG, PVR, PD-1 and PD-L1, determining the percentage of cellsin said population that express the protein relative to said isotypecontrol antibody; wherein if the percentage of positive cells is >1% forall 5 receptors, e) administering antibodies to TIGIT, PVRIG, and PD-1to said patient.

In some embodiments, the anti-TIGIT antibody is an antibody chosen fromany anti-TIGIT antibody described herein, including any of thosedescribed in FIG. 3 .

In some embodiments, the anti-PVRIG antibody is an antibody chosen fromany anti-PVRIG antibody described herein, including any of thosedescribed in FIG. 5 and/or FIG. 63 .

In some embodiments, the anti-PD-1 antibody is an antibody chosen fromany anti-PD-1 antibody described herein, including any of thosedescribed in FIG. 7 .

In some embodiments, the TIGIT antibody is CPA.9.086.

In some embodiments, the PD-1 antibody is selected from pembrolizumaband nivolumab.

In some embodiments, the PVRIG antibody is CHA.7.518.1.H4(S241P).

In some embodiments, the present invention provides a method comprising:a) providing a cell population from a tumor sample from a patient; b)staining said population with labeled antibodies that bind: i) PVRIGprotein; ii) TIGIT protein; iii) PVRL2 protein; iv) PD-1 protein; v)PD-L1 protein; and vi) a relevant isotype control for the antibodies ini)-v); c) running fluorescence activated cell sorting (FACS); d) foreach of PVRIG, TIGIT, PVRL2, PD-1 and PD-L1, determining the percentageof cells in said population that express the protein relative to saidisotype control antibody; wherein if the percentage of positive cellsis >1% for all 5 receptors, e) administering antibodies to PVRIG andPD-1 to said patient.

In some embodiments, the anti-TIGIT antibody is an antibody chosen fromany anti-TIGIT antibody described herein, including any of thosedescribed in FIG. 3 .

In some embodiments, the anti-PVRIG antibody is an antibody chosen fromany anti-PVRIG antibody described herein, including any of thosedescribed in FIG. 5 and/or FIG. 63 .

In some embodiments, the anti-PD-1 antibody is an antibody chosen fromany anti-PD-1 antibody described herein, including any of thosedescribed in FIG. 7 .

In some embodiments, the anti-PD-L1 antibody is an antibody chosen fromany anti-PD-L1 antibody described herein, including any of thosedescribed in FIG. 62 .

In some embodiments, the PVRIG antibody is CHA.7.518.1.H4(S241P).

In some embodiments, the PD-1 antibody is selected from pembrolizumaband nivolumab.

In some embodiments, the TIGIT antibody is CPA.9.086.

In some embodiments, the present invention provides a method comprising:a) providing a cell population from a tumor sample from a patient; b)staining said population with labeled antibodies that bind: i) PVRIGprotein; ii) PD-1 protein; iii) PVRL2 protein; iv) TIGIT protein; v) PVRprotein; and vi) a relevant isotype control for the antibodies in i)-v);c) running fluorescence activated cell sorting (FACS); d) for each ofPVRIG, PVRL2, TIGIT and PVR, determining the percentage of cells in saidpopulation that express the protein relative to said isotype controlantibody; wherein if the percentage of positive cells is >1% for all 5receptors, e) administering antibodies to PVRIG and TIGIT to saidpatient.

In some embodiments, the anti-TIGIT antibody is an antibody chosen fromany anti-TIGIT antibody described herein, including any of thosedescribed in FIG. 3 .

In some embodiments, the anti-PVRIG antibody is an antibody chosen fromany anti-PVRIG antibody described herein, including any of thosedescribed in FIG. 5 and/or FIG. 63 .

In some embodiments, the anti-PD-1 antibody is an antibody chosen fromany anti-PD-1 antibody described herein, including any of thosedescribed in FIG. 7 .

In some embodiments, the anti-PD-L1 antibody is an antibody chosen fromany anti-PD-L1 antibody described herein, including any of thosedescribed in FIG. 62 .

In some embodiments, the PVRIG antibody is CHA.7.518.1.H4(S241P).

In some embodiments, the TIGIT antibody is CPA9.086.

In some embodiments, the PD-1 antibody is selected from pembrolizumaband nivolumab.

In some embodiments, the present invention provides a method of treatingcancer in a patient comprising: a) providing a biopsy from said patientcomprising tumor cells; b) measuring the frequency of PD-L1 positivetumor cells or immune cells in said biopsy; c) if said frequency ofPD-L1 positive tumor cells or immune cells is greater than 1% comparedto staining the same tumor cells with a relevant isotype controlantibody for the antibodies used, administering a triple combinationtherapy comprising an anti-TIGIT antibody, an anti-PVRIG antibody and ananti-PD-L1 antibody; and d) if said frequency of PD-L1 positive tumorcells or immune cells is less than 1% compared to staining the sametumor cells with a relevant isotype control antibody for the antibodiesused, administering a double combination therapy comprising ananti-TIGIT antibody and an anti-PVRIG antibody.

In some embodiments, the anti-TIGIT antibody is an antibody chosen fromany anti-TIGIT antibody described herein, including any of thosedescribed in FIG. 3 .

In some embodiments, the anti-PVRIG antibody is an antibody chosen fromany anti-PVRIG antibody described herein, including any of thosedescribed in FIG. 5 and/or FIG. 63 .

In some embodiments, the anti-PD-1 antibody is an antibody chosen fromany anti-PD-L1 antibody described herein, including any of thosedescribed in FIG. 62 .

In some embodiments, the anti-TIGIT antibody is an antibody chosen fromat least one of CPA.9.083.H4(S241P), CPA.9.086.H4(S241P),CHA.9.547.7.H4(S241P), and CHA.9.547.13.H4(S241P).

In some embodiments, the anti-PVRIG antibody is an antibody chosen fromat least one of CHA. 7.518.1.H4(S241P) and CHA. 7.538.1.2.H4(S241P).

58. A method according to any one of claims 52 to 57, wherein saidanti-PD-L1 antibody is an antibody chosen from at least one ofatezolizumab, avelumab, and durvalumab.

In some embodiments, the double combination therapy is chosen from theadministration of CPA.9.083.H4(S241P) and CHA.7.518.1.H4(S241P);CPA.9.086.H4(S241P) and CHA.7.518.1.H4(S241P); CHA.9.547.7.H4(S241P) andCHA.7.518.1.H4(S241P); CHA.9.547.13.H4(S241P) and CHA.7.518.1.H4(S241P);CPA.9.083.H4(S241P) and CHA.7.538.1.2.H4(S241P); CPA.9.086.H4(S241P) andCHA.7.538.1.2.H4(S241P); CHA.9.547.7.H4(S241P) andCHA.7.538.1.2.H4(S241P); and CHA.9.547.13.H4(S241P) andCHA.7.538.1.2.H4(S241P).

In some embodiments, the triple combination therapy is chosen from theadministration of CPA.9.083.H4(S241P), atezolizumab andCHA.7.518.1.H4(S241P); CPA.9.086.H4(S241P), atezolizumab andCHA.7.518.1.H4(S241P); CHA.9.547.7.H4(S241P), atezolizumab andCHA.7.518.1.H4(S241P); CHA.9.547.13.H4(S241P), atezolizumab andCHA.7.518.1.H4(S241P); CPA.9.083.H4(S241P), atezolizumab andCHA.7.538.1.2.H4(S241P); CPA.9.086.H4(S241P), atezolizumab andCHA.7.538.1.2.H4(S241P); CHA.9.547.7.H4(S241P), atezolizumab andCHA.7.538.1.2.H4(S241P); CHA.9.547.13.H4(S241P), atezolizumab andCHA.7.538.1.2.H4(S241P); CPA.9.083.H4(S241P), avelumab andCHA.7.518.1.H4(S241P); CPA.9.086.H4(S241P), avelumab andCHA.7.518.1.H4(S241P); CHA.9.547.7.H4(S241P), avelumab andCHA.7.518.1.H4(S241P); CHA.9.547.13.H4(S241P), avelumab andCHA.7.518.1.H4(S241P); CPA.9.083.H4(S241P), avelumab andCHA.7.538.1.2.H4(S241P); CPA.9.086.H4(S241P), avelumab andCHA.7.538.1.2.H4(S241P); CHA.9.547.7.H4(S241P, avelumab andCHA.7.538.1.2.H4(S241P); and CHA.9.547.13.H4(S241P), avelumab andCHA.7.538.1.2.H4(S241P); CPA.9.083.H4(S241P), durvalumab andCHA.7.518.1.H4(S241P); CPA.9.086.H4(S241P), durvalumab andCHA.7.518.1.H4(S241P); CHA.9.547.7.H4(S241P), durvalumab andCHA.7.518.1.H4(S241P); CHA.9.547.13.H4(S241P), durvalumab andCHA.7.518.1.H4(S241P); CPA.9.083.H4(S241P), durvalumab andCHA.7.538.1.2.H4(S241P); CPA.9.086.H4(S241P), durvalumab andCHA.7.538.1.2.H4(S241P); CHA.9.547.7.H4(S241P, durvalumab andCHA.7.538.1.2.H4(S241P); and CHA.9.547.13.H4(S241P), durvalumab andCHA.7.538.1.2.H4(S241P).

In some embodiments, the antibodies are administered simultaneously.

In some embodiments, the antibodies are administered sequentially.

In some embodiments, the cancer is selected from the group consisting ofprostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer,endometrial cancer, breast cancer, triple negative breast cancer,pancreatic cancer, stomach (gastric) cancer, cervical cancer, head andneck cancer, thyroid cancer, testis cancer, urothelial cancer, lungcancer (small cell lung, non-small cell lung), melanoma, non melanomaskin cancer (squamous and basal cell carcinoma), glioma, renal cancer(RCC), lymphoma (NHL or HL), Acute myeloid leukemia (AML), T cell AcuteLymphoblastic Leukemia (T-ALL), Diffuse Large B cell lymphoma,testicular germ cell tumors, mesothelioma, esophageal cancer, MerkelCells cancer, MSI-high cancer, KRAS mutant tumors, adult T-cellleukemia/lymphoma, and Myelodysplastic syndromes (MDS).

In some embodiments, the cancer is selected from the group consisting oftriple negative breast cancer, stomach (gastric) cancer, lung cancer(small cell lung, non-small cell lung), and Merkel Cells cancer,MSI-high cancer, KRAS mutant tumors, adult T-cell leukemia/lymphoma, andMyelodysplastic syndromes (MDS).

In some embodiments, the present invention provides a method of treatingcancer in a patient comprising administering a triple combinationtherapy comprising an anti-TIGIT antibody, an anti-PVRIG antibody, andan anti-PD-L1 antibody.

In some embodiments, the anti-TIGIT antibody is an antibody chosen fromat least oneof CPA.9.083.H4(S241P), CPA.9.086.H4(S241P),CHA.9.547.7.H4(S241P), and CHA.9.547.13.H4(S241P).

In some embodiments, the anti-PVRIG antibody is an antibody chosen fromat least one of CHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P).

In some embodiments, the anti-PD-L1 antibody is an antibody selectedfrom the group consisting of atezolizumab, avelumab, and durvalumab.

In some embodiments, the triple combination therapy comprises theadministration of an anti-PD-L1 antibody in combination with adouble-combination therapy chosen from the administration ofCPA.9.083.H4(S241P) and CHA.7.518.1.H4(S241P); CPA.9.086.H4(S241P) andCHA.7.518.1.H4(S241P); CHA.9.547.7.H4(S241P) and CHA.7.518.1.H4(S241P);CHA.9.547.13.H4(S241P) and CHA.7.518.1.H4(S241P); CPA.9.083.H4(S241P)and CHA.7.538.1.2.H4(S241P); CPA.9.086.H4(S241P) andCHA.7.538.1.2.H4(S241P); CHA.9.547.7.H4(S241P) andCHA.7.538.1.2.H4(S241P); and CHA.9.547.13.H4(S241P) andCHA.7.538.1.2.H4(S241P).

In some embodiments, the triple combination therapy is chosen from theadministration of CPA.9.083.H4(S241P), atezolizumab andCHA.7.518.1.H4(S241P); CPA.9.086.H4(S241P), atezolizumab andCHA.7.518.1.H4(S241P); CHA.9.547.7.H4(S241P), atezolizumab andCHA.7.518.1.H4(S241P); CHA.9.547.13.H4(S241P), atezolizumab andCHA.7.518.1.H4(S241P); CPA.9.083.H4(S241P), atezolizumab andCHA.7.538.1.2.H4(S241P); CPA.9.086.H4(S241P), atezolizumab andCHA.7.538.1.2.H4(S241P); CHA.9.547.7.H4(S241P), atezolizumab andCHA.7.538.1.2.H4(S241P); CHA.9.547.13.H4(S241P), atezolizumab andCHA.7.538.1.2.H4(S241P); CPA.9.083.H4(S241P), avelumab andCHA.7.518.1.H4(S241P); CPA.9.086.H4(S241P), avelumab andCHA.7.518.1.H4(S241P); CHA.9.547.7.H4(S241P), avelumab andCHA.7.518.1.H4(S241P); CHA.9.547.13.H4(S241P), avelumab andCHA.7.518.1.H4(S241P); CPA.9.083.H4(S241P), avelumab andCHA.7.538.1.2.H4(S241P); CPA.9.086.H4(S241P), avelumab andCHA.7.538.1.2.H4(S241P); CHA.9.547.7.H4(S241P, avelumab andCHA.7.538.1.2.H4(S241P); and CHA.9.547.13.H4(S241P), avelumab andCHA.7.538.1.2.H4(S241P); CPA.9.083.H4(S241P), durvalumab andCHA.7.518.1.H4(S241P); CPA.9.086.H4(S241P), durvalumab andCHA.7.518.1.H4(S241P); CHA.9.547.7.H4(S241P), durvalumab andCHA.7.518.1.H4(S241P); CHA.9.547.13.H4(S241P), durvalumab andCHA.7.518.1.H4(S241P); CPA.9.083.H4(S241P), durvalumab andCHA.7.538.1.2.H4(S241P); CPA.9.086.H4(S241P), durvalumab andCHA.7.538.1.2.H4(S241P); CHA.9.547.7.H4(S241P, durvalumab andCHA.7.538.1.2.H4(S241P); and CHA.9.547.13.H4(S241P), durvalumab andCHA.7.538.1.2.H4(S241P).

In some embodiments, the antibodies are administered simultaneously.

In some embodiments, the antibodies are administered sequentially.

In some embodiments, the cancer is selected from the group consisting ofprostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer,endometrial cancer, breast cancer, triple negative breast cancer,pancreatic cancer, stomach (gastric) cancer, cervical cancer, head andneck cancer, thyroid cancer, testis cancer, urothelial cancer, lungcancer (small cell lung, non-small cell lung), melanoma, non melanomaskin cancer (squamous and basal cell carcinoma), glioma, renal cancer(RCC), lymphoma (NHL or HL), Acute myeloid leukemia (AML), T cell AcuteLymphoblastic Leukemia (T-ALL), Diffuse Large B cell lymphoma,testicular germ cell tumors, mesothelioma, esophageal cancer, MerkelCells cancer, MSI-high cancer, KRAS mutant tumors, adult T-cellleukemia/lymphoma, and Myelodysplastic syndromes (MDS).

In some embodiments, the cancer is selected from the group consisting oftriple negative breast cancer, stomach (gastric) cancer, lung cancer(small cell lung, non-small cell lung), and Merkel Cells cancer,MSI-high cancer, KRAS mutant tumors, adult T-cell leukemia/lymphoma, andMyelodysplastic syndromes (MDS).

III. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-FIG. 1B depicts the amino acid sequences of the constant domainsof human IgG1 (with some useful amino acid substitutions), IgG2, IgG3,IgG4, IgG4 with a hinge variant that finds particular use in the presentinvention, and the constant domains of the kappa and lambda lightchains.

FIG. 2 depicts the sequence of human and cynomolgus macaque (referred toas cyno) TIGIT, PVRIG and PD-1 proteins.

FIG. 3A-FIG. 3PPPP depicts the sequences of four anti-TIGIT antibodiesthat block the interaction of TIGIT and PVR, CPA.9.083.H4(S241P),CPA.9.086.H4(S241P), CHA.9.547.7.H4(S241P) and CHA.9.547.13.H4(S241P),as well as benchmark antibodies, BM26 and BM29.,and numerous otheranti-TIGIT antibodies.

FIG. 4A-FIG. 4C shows the FACS KD results of anti-TIGIT antibodies(CPA.9.083.H4(S241P), CPA.9.086.H4(S241P), CHA.9.547.7.H4(S241P), andCHA.9.547.13.H4(S241P), as well as benchmark antibodies, BM26 and BM29,binding to human (FIG. 4A-FIG. 4B), cynomolgus (FIG. 4C-FIG. 4D), andmouse (FIG. 4E) TIGIT over-expressing Expi293 HEK cells.

FIG. 5A-FIG. 5F depicts the sequences of two anti-PVRIG antibodies,CHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P). Other PVRIGantibodies are provided in FIG. 63 .

FIG. 6A-FIG. 6B shows the binding of CHA.7.518.1.H4(S241P) to PVRIG byflow cytometry. (A) Binding to PVRIG over-expressing HEK293 cells.CHA.7.518.1.H4(S241P) binds human and cynomolgus PVRIG over-expressingHEK293 cells but not the mouse PVRIG over-expressing or parental HEK293cells. (B) Binding of CHA.7.518.1.H4(S241P) to Jurkat cells. Specificbinding is observed for CHA.7.518.1.H4(S241P) but not for an irrelevantisotype control antibody. Dissociation constant (KD) forCHA.7.518.1.H4(S241P) binding to targets expressed on cells are listedin the table.

FIG. 7A-FIG. 7F depicts the sequences of two anti-PD-1 antibodies.

FIG. 8A-FIG. 8B shows the expression of PVRIG, TIGIT, PD1 on CMVpp65reactive T cells, as described in experiments of Example 1. (A) Gatingstrategy for detection of tetramer-stained CMV-CTLs. The gatinghierarchy and tetramer positive cells in three donors are shown. Thelymphocytes were gated in the FS/SS quadrant (upper left) followed bythe selection of singlets, followed by the removal of CD14-CD19-CD56−cells, followed by CD3+ CD8+ positive cells. Within the CD3+ CD8+positive population, the percentage of cells that bind each tetramer isdetermined in individual donors. Staining results using the HLA-A*02:01CMV tetramer are shown. (B) The expression of PVRIG, TIGIT and PD-1 onthe CMVpp65 reactive T cells expanded from 3 donors is shown.

FIG. 9A-FIG. 9D shows the kinetics of PVRIG, TIGIT and PD-1 expressionon CD8 + CMV+T cells, as described in experiments of Example 1. (A)Percent pp65 tetramer positive of CD8 T cells after 0, 72, 144, 216 and288 hours of stimulation with IL-2, IL-7 and CMV pp65 peptide is shown.(B) TIGIT, (C) CHA.7.518.1.H4(S241P), (D) PD-1 expression on CMVpp65reactive CD8 T-cells at distinct time points after stimulation. (n=3)

FIG. 10 shows the expression of PVRL2, PVR, PDL1 and HLA-A2 on theColo205 and Panc.04.05 cells was evaluated by flow cytometry, asdescribed in experiments of Example 1. The number in the upper righthand corner denotes the percentage of the ligand (PVRL2, PVR, PDL1) orHLA-A2 expressed on the tumor cell lines in comparison to the isotypecontrol antibody.

FIG. 11A-FIG. 11G shows the effect of inhibitory receptor blockade onCMVpp65 reactive CD8 T cells in co-culture with cancer cell lines, asdescribed in experiments of Example 1. CMVpp65 reactive T cells for 2donors (Donor 4 and Donor 156 were co-cultured with 0.03 ug/ml CMVpp65peptide loaded Panc.04.05 or Colo205 for 24 hr in the presence of 10ug/ml CHA.7.518.1.H4(S241P), anti-TIGIT, anti-PD-1, or isotype controleither alone or in combination. (A) CHA.7.518.1.H4(S241P), anti-TIGIT oranti-PD1 antibodies tested alone, in dual combination, or in triplecombination. (B) CHA.7.518.1.H4(S241P) and anti-TIGIT antibodies testedalone or in combination in Donor 4 and Donor 156. C)CHA.7.518.1.H4(S241P) and anti-TIGIT antibodies tested alone or incombination in Donor 4 and Donor 156. (D) CHA.7.518.1.H4(S241P) andanti-PD1 antibodies tested alone or in combination in Donor 4 and Donor156. E) CHA.7.518.1.H4(S241P) and anti-PD1 antibodies tested alone or incombination in Donor 4 and Donor 156. (F) Anti-TIGIT and anti-PD1antibodies tested alone or in combination. G) Anti-TIGIT and anti-PD1antibodies tested alone or in combination. Conditioned media wereassayed for cytokine secretion. The bar graphs show the average+standard deviation for IFN-γ, with each dot representing a technicalreplicate. Data are representative of n>2 experiments.

FIG. 12A-FIG. 12C shows the expression of PVRIG on cells fromdissociated tumors, as described in experiments of Example 2. (A)Samples were grouped based on the tumor type as defined by the pathologyreport. For each sample, expression of PVRIG is shown on CD4+ T cells,CD8+ T cells, CD4-CD8− T cells, and on NK cells. Each dot within acolumn represents an individual sample. Samples with a MFIr value above1 denotes expression of PVRIG was detected. The median is depicted bythe middle line and the upper and lower quartiles are depicted by thegrey space above and below the median line. (B) Across all tumor samplesexamined, the expression of PVRIG on CD4+ T cells, CD8+ T cells,CD4-CD8− T cells, and on NK cells is shown. The median is depicted bythe middle line and the upper and lower quartiles are depicted by thelight and dark grey space above and below the median line. The whiskersdepict 1.5 times the interquartile range. (C) Representative FACShistograms for PVRIG (blue) compared to isotype control (red) are shownfor 4 cell subsets isolated from a lung and kidney tumor.

FIG. 13 shows the correlation analysis of PD1, TIGIT, and PVRIGexpression on CD4+ and CD8+ T cells from dissociated endometrial tumors,as described in experiments of Example 2. For each endometrial sample, aMFIr was calculated for PVRIG, PD1, and TIGIT on CD4 and CD8 T cells. ASpearman's correlation analysis was performed and a r2 and p valuereported.

FIG. 14 shows the co-expression analysis of PD1, TIGIT, and PVRIGexpression on CD8+ T cells from a dissociated lung and kidney cancersample, as described in experiments of Example 2.

FIG. 15A-FIG. 15B shows a comparison of PVRIG expression on T cells fromdissociated tumors with matched NAT, as described in experiments ofExample 2. (A) Matched tumor and normal adjacent tissues fromcolon/stomach/rectal, endometrium/uterine, kidney, lung, and ovariantumors were assessed for PVRIG expression on CD4 and CD8 T cells. Eachline represents a matched donor. A paired Student's t-test was performedon all samples comparing NAT vs tumor expression of PVRIG on CD4 and CD8T cells. (B) PVRIG fold change (in NAT vs tumor) is plotted vs PD1 foldchange for CD4 and CD8 T cells. A spearman's correlation analysis wasdone and r value and p value is shown.

FIG. 16 shows the expression of PVRL2 on immune and non-immune subsetsfrom all dissociated tumor samples, as described in experiments ofExample 2. The expression of PVRL2 on various cell subsets derived fromtumors is shown. MFIr values above 1 denote expression of PVRL2 wasdetected. The median is depicted by the middle line and the upper andlower quartiles are depicted by the light and dark grey space above andbelow the median line. The whiskers depict 1.5 times the interquartilerange.

FIG. 17 shows the expression of PVRL2 on non-immune subsets fromdissociated tumors, as described in experiments of Example 2. Sampleswere grouped based on the tumor type as defined by the pathology report.For each sample, expression of PVRL2 is shown on CD45- non-immune cells.Each dot represents an individual sample. The median is depicted by themiddle line and the upper and lower quartiles are depicted by the greyspace above and below the median line.

FIG. 18 shows the expression of PVRL2 on myeloid cell subsets fromdissociated tumors, as described in experiments of Example 2. Sampleswere grouped based on the tumor type as defined by the pathology report.For each sample, expression of PVRL2 is shown on myeloid cells whichinclude monocytes, mDC, and pDC populations. Each dot represents anindividual sample. The median is depicted by the middle line and theupper and lower quartiles are depicted by the grey space above and belowthe median line.

FIG. 19A-FIG. 19B shows a comparison of PVRL2 expression on monocytesand CD45− tumor cells from dissociated tumors and matched NAT, asdescribed in experiments of Example 2. (A) Matched tumor and normaladjacent tissues from colon/stomach/rectal, endometrium/uterine, kidney,lung, and ovarian tumors were assessed for PVRL2 expression on CD45−cells and on monocytes. Each line represents a matched donor. A pairedStudent's t-test was performed on all samples comparing NAT vs tumorexpression of PVRL2 on CD45− cells and on monocytes cells. (B) PVRL2fold change (in NAT vs tumor) is plotted vs PD-L1 fold change for CD45−cells and for monocytes. A spearman's correlation analysis was done andr value and p value is shown.

FIG. 20 depicts the co-expression of PVRIG on T cells with PVRL2 onmonocytes and CD45− cells in tumor tissues, as described in experimentsof Example 2. From the same sample, the expression of PVRIG on CD8 Tcells and PVRL2 on monocytes or CD45− cells was plotted. Tumor typeswere grouped and each dot represents an individual tumor. Referencelines were drawn at MFIr value of 2.

FIG. 21A-FIG. 21E depicts the expression of PVRL2 and PD-L1 in colon,skin, and breast cancers, as described in experiments of Example 3.

FIG. 22A-FIG. 22B depicts the expression of PVRL2 in PD-L1 negative andPD-L1 positive tumors, as described in experiments of Example 3. Basedon PD-L1 staining, tumors were categorized as PD-L1 Negative (Nostaining of PD-L1 observed in either duplicate cores for each tumor) orPD-L1 Positive (Positive staining observed in both duplicate cores foreach tumor. A) Expression of PVRL2 was analyzed and shown for eachcancer type. B) Of the PD-L1 negative tumors, the number of PVRL2expressing samples (PVRL2 partial positive or greater/total samples) foreach cancer type is shown.

FIG. 23A-FIG. 23B shows the expression of PVRL2 and PD-L1 at theinvasive front of the tumor, as described in experiments of Example 3.A. In this tumor sample, PVRL2 was expressed on both immune cells andtumor cells at the invasive front, as delineated by the blue and redlines. B) In this tumor sample, PD-L1 was expressed in the immunecompartment.

FIG. 24A-FIG. 24C shows antitumor responses of mono, dual and triplecombination antibody treatments in the CT26 tumor model, as described inexperiments of Example 4. Groups of 10-15 Balb/c mice weresubcutaneously injected with 5×10⁵ CT26 cells. Mice were treated ×2weekly for 3 weeks, starting at day 7 post inoculation with thedesignated antibody combination. A) Tumor volumes of all tested groupswere measured x2 weekly, including positive control group (anti-PDL-1 +anti-CTLA-4 antibodies). The TGIs and p-values of the triple combinationgroup compared to the indicated groups summarized in the table. B)Survival proportions of assigned groups. C) Spider plots showingindividuals response over treatment groups, while PR indicates partialresponders with tumor size not exceeding 1000 mm³.

FIG. 25A -FIG. 25D depicts expression profiles for PVRIG and PVRL2, aswell as PD-L1 in various human tumors, as described in experiments ofExamples 2 and 3.

FIG. 26 depicts in vivo data regarding the use of an anti-PVRIG antibodyin TIGIT−/− mice or the combination of anti-PVRIG and anti-PD-1antibodies in wild type Balb/c mice to reduce syngeneic tumor growth, asdescribed in experiments of Example 4.

FIG. 27A-FIG. 27H. PVRIG is expressed highest on cytotoxic lymphocytesubsets from human cancer. A) Expression of PVRIG on leukocyte cellsubsets from 5-8 healthy donor PBMCs is shown. PVRIG expression isdefined as the ratio of PVRIG MFI relative to isotype control MFI. B)Expression of PVRIG, TIGIT, CD96, and PD-1 on peripheral blood Tregs ascompared to CD8 T cell subsets from 5 healthy donor PBMCs is shown. C)CMV pp65 specific T cells from 3 healthy donors were expanded in vitrowith pp65 (495-503) peptide, IL-2 and IL-7 for up to 7 days. Expressionof TIGIT (blue) and PVRIG (black) on HLA-A2/pp65 (495-503) tetramerpositive cells is shown. D) Human T cells were cultured with allogeneicDCs and expression of TIGIT and PVRIG shown on CD4+ T cells on day 0, 1,2, and 7 post activation. E) Representative FACS plots showingexpression of PVRIG (blue) compared to isotype control (red) on TILS(CD4 T cells, CD8 T cells, and NK cells) from a representative lung andkidney cancer. F) Co-expression of PVRIG, TIGIT, and PD-1 on CD4 and CD8TILS from a lung cancer sample is shown. G) Expression of PVRIG on CD8⁺and CD4⁺ TILS from dissociated human tumors of various cancer types isshown. Each dot represents a distinct tumor from an individual patient.H) Relative expression on CD8 TILs vs Treg TILS for PVRIG, TIGIT, andPD-1 from endometrial, kidney, and lung tumors was assessed. For eachtumor, the fold expression on CD8 TILS was normalized to fold expressionon Treg TILS and plotted. For A, B, C, G, and H, mean+SEM is shown bythe error bars.

FIG. 28A-FIG. 28F. PVRL2 expression is enhanced in the tumormicroenvironment. A) PVRL2 expression was assessed by IHC on lung,ovarian/endometrial, breast, colon, and kidney tumors. For each tumor, 2cores were assessed by 2 independent observers. Representative stainingfor each descriptor is shown in FIG. B) A representative melanoma tumorshowing PVRL2 expression on tumor cells and in the immune cells in thestroma is shown. C) PVRL2 expression from dissociated tumors wasexamined by FACS on CD45⁻, CD14⁺ TAMs, and CD14⁻CD33^(hi) mDC cellsubsets. Mean+SEM is shown for each cancer type. D) Representative FACSplots for PVRL2 expression (blue) as compared to IgG (red) are shown fora lung cancer. E) For tumor samples where we were able to assess bothPVRIG and PVRL2 expression, PVRIG expression on CD8 T cells is plottedversus PVRL2 expression on CD14⁺ TAMS for each tumor. Each dotrepresents an individual tumor sample. Red line represents a 2 foldexpression of PVRIG or PVRL2 compared to IgG.

FIG. 29A-FIG. 29E. Distinct regulation of PVRL2 and PD-L1 on tumorcells. A) Expression of PD-L1 and PVRL2 was assessed by IHC on serialsections. Expression of PVRL2 on PD-L1 negative (left) and PD-L1positive (tumors) is shown. PD-L1 negative tumors were defined as nostaining observed on duplicate cores for a given tumor. PD-L1 positivestaining was defined as at least partial positive on both duplicatecores of a give tumor. The number of PVRL2 positive tumors from PD-L1positive and PD-L1 negative tumors is shown in the table(positive/total). B, C) Representative expression of a PVRL2⁺PD-L1⁻endometrial (B) tumor and a PVRL2⁺PD-L1⁻ lung (C) tumor. D) ImmatureBM-DCs were cultured with the indicated stimuli and PVR, PVRL2, PD-L1expression assessed by FACS on day 2 of culture. For each condition,expression was normalized to media only control condition. E) Expressionof PVR, PVRL2, and PD-L1 on HT-29 cells treated with IFN-γ or mediaalone is shown. PD-L1 or PVRL2 is shown in blue and IgG isotype controlstaining is shown in red.

FIG. 30A-FIG. 30I. CHA.7.518.1.H4(S241P) is a high affinity antibodythat enhances T cell activation. A) Binding of CHA.7.518.1.H4(S241P) orIgG isotype control to HEK293 PVRIG or HEK293 parental cells by FACS isshown. FACS KD values are shown for the binding of CHA.7.518.1.H4(S241P)to HEK293 hPVRIG, HEK293 cPVRIG, and Jurkat cells. B)CHA.7.518.1.H4(S241P) disrupts the binding of PVRL2 Fc to HEK293 cellsectopically expressing PVRIG. Mean+Std Dev of triplicate values isshown. C) CHA.7.518.1.H4(S241P) blocks the binding of PVRIG Fc to HEK293cells that endogenously express PVRL2. D) Human CD4 T cells wereco-cultured with aAPC CHO cells expressing a cell surface bound anti-CD3antibody and hPVRL2 in the presence of 10 μg/ml anti-PVRIG antibody andhuman IgG isotype control antibodies. The effect of anti-PVRIG Ab onproliferation of CD4 T cells isolated from 11 different donors is shown.Bars depicted mean+SEM. E) gp100 specific T cell lines (TIL-209,TIL-463) were co-cultured with CHO cells engineered to express HLA-A2and PVRL2 along with 10 μg/ml anti-PVRIG or IgG isotype controlantibody. IFN-γ and TNF-α production was tested at 24 hours postco-culture. Mean+Std Dev of triplicate values is shown. Percent changein IFN-γ and TNF-α for each condition relative to isotype control isdepicted by the number above each bar. F) Expression of PVR, PVRL2, andPD-L1 (red) relative to IgG (blue) on MEL624, Colo205, and Panc.05.04cells is shown. For the T cells, expression of PVRIG, TIGIT, and PD-1(red) relative to IgG (blue) on TIL-209 and TIL-463 gp100 specific Tcells, and on CMVpp65 specific T cells is shown. To expand CMVpp65reactive T cells, PBMCs were cultured with pp65 (495-503) peptide, IL-2,and IL-7 for 10 days. Expression of PVRIG, TIGIT, PD-1 is shown onHLA-A2/pp65 tetramer positive cells. G) gp100 specific T cells (TIL-209,TIL-463) expanded from TILS derived from melanoma tumors wereco-cultured with MEL624 cells in the presence of 10 μg/ml of theindicated antibodies. IFN-γ concentration in the conditioned media wasdetermined at 24 hrs. H, I) Expanded CMVpp65 specific T cells wereco-cultured with Colo205 and Panc.05.04 cells, CMVpp65 peptide, and theindicated antibodies at 10 μg/ml. IFN-y concentration in the conditionedmedia was determined at 24 hrs. For E, G, H, I, average+Std Dev oftriplicates is shown. Percent change in IFN-γ for each conditionrelative to isotype control is depicted by the number above each bar.

FIG. 31A-FIG. 31E. PVRIG deficient mice have increased T cell function.A) RNA expression of PVRIG as measured by qRT-PCR from purified mouseimmune cell subsets was assessed. Relative expression to housekeepingwas determined by ΔCt method. B) pmel CD8⁺ TCR transgenic T cells wereactivated with gp100 (25-33) and PVRIG and TIGIT RNA transcript levelsassessed by qRT-PCR at the indicated time points. Graph shows mean+SEMof results from 5 different experiments. C) Spleens were harvested fromPVRIG^(−/−) and WT littermates and analyzed by flow cytometry forexpression of PVRIG on NK, CD4⁺ and CD8⁺ T cells (“Resting” cells). Inaddition, CD3⁺ T cells were isolated from splenocytes and activated for11 days with anti-CD³/_(a)nti-CD28 beads. Following the activation,PVRIG expression on CD4⁺ and CD8⁺ T cells (“activated” cells) wasanalyzed by flow cytometry. Each dot represents cells derived from anindividual mouse. D) WT and PVRIG^(−/−) derived splenocytes were labeledwith Cell Proliferation Dye eFluor450 and were cultured in the presenceof Control-Fc (mouse IgG2a) or with mouse PVRL2 Fc. After 4 d ofculture, cell division was analyzed by flow cytometry. RepresentativeFACS plots from an experiment (left) and the summary of percentageinhibition by PVRL2 Fc (defined as % proliferation Control-Fc subtractedfrom % proliferation PVRL2 Fc) 3 independent experiments (right) arepresented. * indicate p-value<0.05, paired student's t-test for thechange in proliferation in the presence of PVRL2-FC relative toproliferation in the presence of protein control in WT versusPVRIG^(−/−) T cells E) pmel CD8+ T cells derived from pmel PVRIG^(−/−)or pmel PVRIG WT mice were activated for 11 days with their cognatepeptide and IL2. Activated pmel CD8⁺ cells were then co-cultured withB16-Db/ 100 cells for 18 hours and following the co-culture wereevaluated for CD107 expression and for cytokine production. Fourindependent experiments are presented as indicated by each paired dot. *indicate p-value<0.05, Student's t-test comparing PVRIG^(−/−) versus WT.

FIG. 32A-FIG. 32H. PVRIG deficiency results in reduced tumor growth andincreased CD8 effector T cell mechanism. (A) C57BL/6 WT or PVRIG^(−/−)mice were subcutaneously injected with 5×10⁵ MC38 cells. Tumor volumeswere measured ×2 weekly. * indicate p-value<0.05 for WT mice versusPVRIG^(−/−) mice (ANOVA). (B) Individual tumor growth curves are shown.One representative experiment out of 2 performed is shown. (C) C57BL/6WT or PVRIG^(−/−) mice were subcutaneously injected with 5×10⁵ MC38cells. At day 14 post- inoculation, mice were treated with anti-PD-L1,×2 weekly for 2 weeks. Tumor volumes were measured ×2 weekly.p-value=0.052 for WT mice versus PVRIG^(−/−) mice, both treated withanti-PD-L1. (D) Individual tumor growth curves are shown. Onerepresentative experiment out of 2 performed is shown. (E) Frequency ofCD8⁺ IFN-γ⁺TNF-α⁺ effector cells in tumor-draining lymph nodes from 4treatment groups on day 18 is shown. (F) Total number of CD8+IFN-γ+TNF-α+ effector cells per mg tumor tissue on day 18 is shown.(G-H) Total TILS score and Cytotoxic T cells score relative to TILs,derived from nSolver 3.0 advanced analysis of the mouse pan-cancerimmune codeset panel (Nanostring Technologies, Seattle, Wash.) run onCD45+ enriched cells from MC38 day 18 TILs isolated from 2 treatmentgroups per wild-type and PVRIG deficient mice.

FIG. 33A-FIG. 33F. Antagonistic anti-PVRIG antibodies synergisticallyinhibit tumor grown in combination of PD-1 inhibitors or TIGIT geneticdeficiency. A) Binding of mPVRL2 Fc fusion protein to mPVRIG HEK293engineered cells that were pre-incubated with serial dilutions ofanti-mPVRIG mAb or IgG isotype control Ab is shown. B) BALB/c mice weresubcutaneously injected with 5×10⁵ CT26 cells. On day 14 postinoculation, mice were sacrificed and spleen, draining lymph nodes andtumors were harvested. Cells were analyzed by flow cytometry forexpression of PVRIG on CD3⁺CD4⁺ T cells, CD3⁺CD8⁺ T cells, CD3⁻CD49b⁺NKcells, CD11b⁺Gr-1⁺ Myeloid-Derived-Suppressor Cells (MDSC) andCD11b⁺F4/80⁺ macrophages. C, D) BALB/c mice were subcutaneously injectedwith 5×10⁵ CT26 cells. At day 7 post inoculation mice were treated withanti-PD-L1 and/or anti-PVRIG Ab, 2× weekly for 3 weeks (arrows indicateAb treatment). C) Tumor volumes are shown. *** indicate p-value<0.001(ANOVA) for anti-PD-L1+Rat IgG2b compared to anti-PD-L1+aPVRIG treatedgroups. Arrows indicate when antibodies were dosed. D. Survival analysisof complete responder's mice. * indicate p value<0.05 (Log-rank test)for anti-PD-L1+ Rat IgG2b compared to anti-PD-L1 + anti-PVRIG treatedgroups. One representative study of 3 studies are shown. E. C57BL/6 orTIGIT^(−/−) mice were subcutaneously injected with 1×10⁵ B16/Db-hmgp100cells. Mice were treated 2× weekly for 3 weeks with the designated mAbstarting on the day of inoculation (day 0). E. Tumor volumes weremeasured 2× weekly and average+SEM is shown. Tumor growth inhibition asmeasured at indicated days compared to control WT+mIgG1 isotype control.*** indicate p-value<0.001 for TIGIT^(−/−)+aPVRIG compared to WT+mIgG1isotype control. Arrows indicate when antibodies were dosed. F.Individual tumor growth curves for each mouse is shown. Onerepresentative experiment out of 2 performed is shown.

FIG. 34A-FIG. 34F . PVRIG is expressed on T and NK cells of TILS inhuman cancer. A) Expression of PVRIG, TIGIT, CD96, and PD-1 on CD4 Tcell subsets from healthy donor PBMCs is shown. Mean+SEM is shown. B)Human T cells were co-cultured with allogeneic PBMCs and expression ofPVRIG protein on CD4 and CD8 T cells shown (top). C) Tumors weredissociated and single cells were activated with anti-CD3 and anti-CD28.Expression of PVRIG (blue) relative to IgG isotype control (red) wasassessed on day 0 (directly ex vivo) and day 5 post activation. D)Expression of PVRIG on NK cells from dissociated human tumors is shown.Each dot represents a distinct tumor from an individual patient.Mean+95% confidence internal is shown. D) Dissociated tumor cells wereactivated with anti-CD3 and anti-CD28 beads for 5 days. Expression ofPVRIG (blue) relative to IgG control (red) on CD4 and CD8 T cells on day0 directly ex vivo and on day 5 post activation is shown for 2dissociated tumor samples. E) Expression of PVRIG was assessed on CD4and CD8 T cells from dissociated tumors and from dissociateddonor-matched normal adjacent tissue. Each line represents matchedtissues obtained from an individual patient. A paired student's t-testwas performed. F) A correlation analysis of the magnitude of PVRIG,TIGIT, and PD-1 fold expression relative to IgG isotype control on CD4and CD8 T cells from tumors is shown. Each dot represents an individualtumor sample. A Spearman's correlation coefficient and p value areshown.

FIG. 35A-FIG. 35F. Expression of PVRL2 is enhanced in colon, skin, andbreast cancers. A) Photomicrographs showing the binding of Sigma antihuman PVRL2 antibody to FFPE sections of positive cells, CHO-S humanPVRL2 (right) compare to negative cells, CHO-S (left), following antigenretrieval at pH9. B) Anti-PVRL2 antibody was tested on a panel of PVRL2⁺(HT29, MCF7, PC3, PANC1, RT4, NCI-H1573) and PVRL2⁻ (Jurkat, OPM2,Daudi, CA46) cell lines. C-F) Example expression of PVRL2 in lung normaland cancer tissues. C) Normal tissue showing no staining. D) LungAdenocarcinoma showing partial positive staining. E) Lung adenocarcinomashowing positive staining. F) Lung adenocarcinoma showing strongpositive staining.

FIG. 36 . PVRL2 is upregulated on TAMs and CD45⁻ cells in the tumor ascompared to normal adjacent tissue. Expression of PVRL2 on CD45⁻ cellsand TAMs from donor matched tumor and normal adjacent tissue is shown. Apaired student's t-test p value is shown.

FIG. 37A- FIG. 37B. PVRIG and PVRL2 are co-expressed in the same tumorsample. PVRIG expression on CD4 T cells (A) and NK cells (B) is plottedagainst PVRL2 expression on TAMS for an individual tumor.

FIG. 38A-FIG. 38D. Activity of CHA.7.518.1.H4(S241P) on human T cells.A) Expression of PVRIG on CD4 T cells activated with CHO cellsexpressing cell surface bound anti-CD3 and PVRL2. B) Expression ofHLA-A2, B-2m, and PVRL2 are shown on CHO-S parental and engineered CHO-Scell lines. Fold expression relative to isotype is depicted by thenumber. C) CHO cells ectopically expressing cell surface bound anti-CD3and PVRL2 were co-cultured with purified CD8 T cells in the presence ofvarying concentrations of anti-PVRIG Ab or relevant IgG control. %Proliferation is shown. Each dot represents an average of triplicatevalues. D) CHO cells ectopically expression HLA-A2/B2m and PVRL2 wereco-cultured with 2 gp100 specific T cell lines (TIL F4, TIL 209) in thepresence of 1 ug/ml gp100 and varying concentrations of anti-PVRIGantibody or relevant IgG control. TNF-α concentrations on day 3 ofco-culture is down. Each value represents an average of triplicates.

FIG. 39A-FIG. 39J. Characterization of mPVRIG binding interactions and asurrogate anti-mPVRIG antibody. A, B) Binding of mPVRIG to mPVRL2 wasassessed by surface plasmon resonance. C) Soluble receptor Fc or controlproteins were incubated in a dose response with immobilized mPVRL2 HISin an ELISA format. Bound receptor Fc is shown. D) Soluble PVRL2 HISprotein was incubated in a dose response with PVRIG Fc or DNAM Fc coatedplates. E) Binding of mPVRIG Fc or control Fc fusion protein to B16-F10cell line transfected with mPVRL2 siRNA, mPVRsRNA, or scrambled siRNAtransfection is shown. F) Affinity characterization of rat anti-mousePVRIG mAb was performed by examining the binding of anti-mPVRIG toHEK293 cells overexpressing mPVRIG. G) Affinity characterization of ratanti-mouse PVRIG mAb was performed by examining the of anti-mPVRIG toD10.G4.1 cell line endogenously expressing mPVRIG vs isotype control ratIgG is shown. H) Binding of anti-mPVRIG to D10.G4.1 cells transfectedwith mouse PVRIG-siRNA (green histogram) vs scr siRNA (orangehistogram). I) Binding of mPVRIG Fc pre-incubated with anti-mPVRIG Ab toB16-F10 cells, which endogenously express PVRL2

FIG. 40A-FIG. 40B. Generation of transgenic PVRIG and TIGIT knockoutmice. The PVRIG conditional knockout and Tigit knockout mouse lines weregenerated by Ozgene Pty Ltd (Bentley Wash., Australia). A) The targetingconstruct in which PVRIG exons 1 to 4 were floxed was electroporatedinto a C57BL/6 ES cell line, Bruce4 (Koentgen et al., Int Immunol 5:957-964, 1993). B) The targeting construct in which the coding region ofTigit exon 1 (including the ATG) and exons 2 and 3 were replaced with anFRT-flanked neo cassette was electroporated into a C57BL/6 ES cell line,Bruce4. Homologous recombinant ES cell clones were identified bySouthern hybridization and injected into goGermline blastocysts(Koentgen et al., genesis 54: 326-333, 2016). Male chimeric mice wereobtained and crossed to C57BL/6J females to establish heterozygousgermline offspring on C57BL/6 background. The germline mice were crossedto a ubiquitous FLP C57BL/6 mouse line to remove the FRT flankedselectable marker cassette and generate the conditional or knockoutalleles (for PVRIG and Tigit, respectively). For PVRIG knockout, micewere further crossed to a ubiquitous Cre C57BL/6 mouse line to removethe loxP flanked exons and generate the knockout allele.

FIG. 41A-FIG. 41I. PVRIG knockout mice are immune-phenotypically similarto wild-type mice. Mice (n=5 per wild-type and PVRIG knockout cohorts)were euthanized prior to venous blood being collected inanti-coagulant-coated tubes and harvesting of organs. Single cells wererecovered from freshly harvested bone marrow, thymus, spleen, cutaneousand mesenteric lymph nodes. Cells were stained withfluorochrome-conjugated surface marker antibodies and acquired on a BDLSR Fortessa flow cytometer. Panels illustrate comparable frequencies ofmyeloid cells (A), dendritic cells (B), B cells (C), T cells (D), CD4 Tcells (E), CD8 T cells (F), and NK cells (G) across lymphoid tissuetypes. (H-I) Whole venous blood was run on a Hemavet 950 veterinaryhematology system to compare differential counts and frequencies ofblood cell subsets from wild-type and PVRIG deficient mice.

FIG. 42 . Increased T cell effector function in PVRIG^(−/−) mice treatedwith anti-PDL1 compared to WT with anti-PD-L1. MC38 tumors wereinoculated into WT or PVRIG^(−/−) mice and were subsequently treatedwith anti-PD-L1 or rat IgG2b isotype control. On day 18, CD45+ tumorinfiltrating lymphocytes were purified from tumors, RNA extracted, andtranscript profiling performed. Several T cell related genes are shown,with each dot representing an individual mouse. Student's t test pvalues are shown.

FIG. 43A-FIG. 43B. Anti-TIGIT and anti-PVRIG antibodies induce tumorcell killing. An in vitro co-culture assay with human CMV-specific CD8+T cells expanded was utilized to assess the effect of the benchmarkanti-TIGIT antibody and CHA.7.518.1.H4(S241P) on antigen-specific tumorcell killing. HLA-A2+ target cell lines used in the assay were theMe1624 (A) and Panc05.04 (B). Synagis hIgG4 is the isotype controlantibody. Luciferase activity in the target cells was measured with theBio-Glo luciferase substrate. Representative data (n≥2) shows thepercent specific killing (mean +/- standard deviation) of Me1624 orPanc05.04 cells after a 16 hour co-culture with human CMV-specific CD8+T cells from three different donors.

FIG. 44 . Dose-dependent tumor cell killing of anti-TIGIT antibodieswith CHA.7.518.1.H4(S241P). An in vitro co-culture assay with humanCMV-specific CD8+ T cells was utilized to assess the effect of twodifferent anti-TIGIT antibodies, BM26 and CPA.9.086 when combined withCHA.7.518.1.H4(S241P) on antigen-specific Me1624 cell killing.Luciferase activity in the target cells was measured with the Bio-Gloluciferase substrate. Representative data (n≥2) shows the percentspecific killing (mean+/−standard deviation) of Me1624 cells after a 16hour co-culture with human CMV-specific CD8+ T cells from one donor.

FIG. 45 . CPA.9.086 CDR sequences, IMGT and Kabat numbering.

FIG. 46 . Anti-TIGIT hIgG4 + CHA.7.518.1.H4(S241P) combination inducestumor cell killing. Co-culture of CMV-reactive CD8+ T cells with Me1624PVR, PVRL2 & luciferase OE Single dose of 10 μg/ml anti-TIGIT Ab and 10μg/ml CHA.7.518.1.H4(S241P) with CMV-reactive donor 4, while dosetitration starting at 0.5 μg/m1 aTIGIT Ab and 10 μg/mlCHA.7.518.1.H4(S241P) with CMV-reactive donor 156.

FIG. 47 . Anti-TIGIT antibodies augment IFN-γ when combined with ananti-PD-1 antibody. An in vitro co-culture assay with human CMV-specificCD8+ T cells was utilized to assess the effect of CPA.9.086 compared tothe benchmark antibodies, BM26 and BM29, on antigen-specific cytokinesecretion in combination with an anti-PD-1 antibody, pembrolizumab. Thetarget cell line used in the assay was the HLA-A2+ pancreaticadenocarcinoma cells, Panc.05.04 that endogenously expresses human PVRand PD-L1. Panc.05.04 cells were pulsed with the CMV pp65 peptide at0.01 μg/ml at 37° C. for 1 hour. Cells were then washed and plated at50,000 cells/well in 96-well round-bottom tissue culture treated plates.Anti-human TIGIT antibodies or the isotype control hIgG4 antibody(anti-Synagis) were added at a concentration of 0.1 μg/ml in combinationwith the anti-PD-1 antibody (hatched bars) or a control hIgG4 isotypeantibody at 10 μg/ml (solid bars). Human CMV-specific CD8+ T cells froma single donor were expanded according to the protocol above. 50,000human CD8+ T cells were added to each well. Co-cultures were incubatedat 37° C. with 5% CO₂ for 24 hours. The amount of human IFN-γ in theco-culture supernatant was measured by flow cytometry using a cytometricbead assay (BD Biosciences).

FIG. 48A-FIG. 48D depicts expression profiling of PVRIG/TIGIT axis intumors; lung and endometrial cancers are high for both PVRIG-PVRL2 andTIGIT-PVR pathway. (A, B) PVRIG and TIGIT expression were analyzed onCD4⁺ and CD8⁺ T cells from dissociated human tumors by FACS. Foldexpression was calculated by dividing the MFI of PVRIG or TIGIT by theMFI of the IgG control. Grey line=No expression detected. Each orangedot is a distinct tumor sample and median of samples shown by the bluebar. C, D) Expression of PVRIG on CD8⁺ T cells vs PVRL2 on CD45⁻ cellsor TIGIT on CD8⁺ T cells vs PVR on CD45⁻ cells is plotted fromdissociated tumors. Each dot represents an individual tumor sample.

FIG. 49A-FIG. 49F depicts expression data for PD-1, PVRIG and TIGIT onCD8 T cells, which shows that PVRIG+TIGIT+PD-1+CD8+TILs are highlyprevalent and have an exhausted profile. A) TILS from human cancers werestained for PD1, PVRIG, and TIGIT expression on CD8 T cells. Thepercentage of cells that express combinations of PD-1, PVRIG, or TIGITon CD8+ T cells was determined by Boolean gating. B) RepresentativePD-1, PVRIG, and TIGIT expression on CD4+ and CD8+ T cells from a lungtumor are shown. C) TILS from human cancers were stained for cellsurface PD1, PVRIG, and TIGIT on CD8+ T cells, permeabilized, andstained for Eomes and T-bet. Within each cell subset, the percentage ofEomes+T-bet- cells are shown. A paired Student's t-test was performedand p values shown. D) Representative FACS plots showing Eomes and T-betexpression on PD-1, PVRIG, or TIGIT expressing CD8 T cells from anovarian and bladder tumor are shown. E) Representative FACS plotsshowing Eomes and T-bet expression on PD-1, PVRIG, or TIGIT expressingCD8 T cells from an ovarian and bladder tumor are shown. F) Percentageof Eomes+T-bet- cells expressing cells based on PD-1, PVRIG, and TIGITexpression was determined. Thus, PVRIG expression correlates withEomes+T-bet- transcription factor expression, a phenotype known to beassociated with T cell exhaustion. Triple positive PVRIG+TIGIT+PD-1+cells were also high in percentage of Eomes+T-bet- cells.

FIG. 50A-FIG. 50C shows that PVRL2 is induced in cancer and expressed inPD-L1⁻ tumors. A) PVRL2 expression was assessed by IHC on lung,ovarian/endometrial, breast, colon, kidney, and skin tumors. Bars depictmean+SEM. For each tumor, 2 cores were assessed by a pathologist andscored based on prevalence and intensity of membranous staining on tumorcells. For each tumor, the average score of 2 cores is shown. B)Expression of PD-L1 and PVRL2 was assessed by IHC on serial sections.Tumors were grouped based on tissue type and expression of PVRL2 onPD-L1 negative and PD-L1 positive is shown. PD-L1 negative tumors weredefined as no membranous staining on tumor or immune cells from eitherduplicate cores for a given tumor. PD-L1 positive staining was definedas membranous staining on at least 1 core of a tumor. Bars depictmean+SEM for each group. C) Representative expression of a PVRL2+PD-L1—endometrioid carcinoma tumor and a PVRL2+PD-L1− lung tumor.

FIG. 51A-FIG. 51B shows that anti-PVRIG/TIGIT/PD-1 synergisticallyincreases T cell function. (A) CMVpp65 CD8 T cells were stained forTIGIT/PD-1/PVRIG expression, and tumor cell lines were stained forPD-L1, HLA-A2, PVR & PVRL2. Representative FACs histograms are shown. B)CMVpp65 specific T cells were co-cultured with Panc0504 & Colo205 cells,CMVpp65 peptide and the indicated antibodies at 10 ug/ml. IFN-yconcentration in the conditioned media was determined at 18 hrs.Percentages above bar graphs is % increase in IFN-y secretion overisotype IgG.

FIG. 52A-FIG. 52C shows that the blockade of the PVRIG-PVRL2 interactionnduces PD-1 and TIGIT expression. CMVpp65 specific T cells from 1-2donors were co-cultured with Panc0504, CMVpp65 peptide, and theindicated antibodies at 10 ug/ml for 18 hours. Cells were then stainedfor FACs and the percentage of cells PD-1, TIGIT, and LAG3 for eachtreatment condition is shown. Representative histograms for eachreceptor is shown. Red=Isotype, Blue=Target expression. A) TIGITexpression was induced by CHA7.518.1.H4(S241P) or anti-PD1 treatment.(B) PD-1 expression was induced by CHA7.518.1.H4(S241P) and/orCPA.9.083.H4(S241P). (C) LAG-3 expression was not induced byCHA7.518.1.H4(S241P), anti-TIGIT, or anti-PD-1.

FIG. 53 . Tumors obtained within 24hrs of surgical resection weredissociated and purified CD3+ TILS co-cultured with MEL624 cellsexpressing surface bound anti-CD3 and the indicated antibodies at 10ug/ml. IFN-γ concentration in the conditioned media was determined at 72hrs. % change in IFN-γ for each condition relative to hIgG4 is shown.

FIG. 54A-FIG. 54C. PVRIG ANTIBODY BLOCKADE OR DEFICIENCY RESULT INREDUCED TUMOR GROWTH PVRIG antibody blockade or deficiency inhibit tumorgrowth. A) Schematic of related signaling pathways. B) BALB/c mice weresubcutaneously injected with 5×105 CT26 cells. At day 7 post inoculationmice were treated with anti-PD-L1 and/or anti-PVRIG antibodies, twiceweekly for 3 weeks. Tumor volumes are shown. n=10 mice per group. Mean+/- SEM is shown. *** Indicates p-value<0.001 (ANOVA with repeatedmeasures) for anti-PD-L1 +Rat IgG2b compared to anti-PD-L1 anti-PVRIGtreated groups. C) C57BL/6 WT or PVRIG−/− mice were subcutaneouslyinjected with 5×105 MC38 cells. n=10 mice per group. Mean+/−SEM isshown. *Indicates p-value<0.05 for WT mice versus PVRIG−/− mice (ANOVAwith repeated measures). Individual tumor growth curves are also shown.Representative data from n=2 experiments.

FIG. 55A-FIG. 55D. EXPRESSION PROFILING OF PVRIG/TIGIT AXIS IN HUMANTUMORS. Lung and endometrial cancers are high for both PVRIG-PVRL2 andTIGIT-PVR pathway. (A, B) PVRIG and TIGIT expression were analyzed onCD4⁺ and CD8⁺ T cells from dissociated human tumors by FACS. Foldexpression was calculated by dividing the MFI of PVRIG or TIGIT by theMFI of the IgG control. Grey line=No expression detected. Each orangedot is a distinct tumor sample and median of samples shown by the bluebar. C, D) Expression of PVRIG on CD8⁺ T cells vs PVRL2 on CD45− cellsor TIGIT on CD8⁺ T cells vs PVR on CD45− cells is plotted fromdissociated human tumors. Each dot represents an individual tumorsample.

FIG. 56A-FIG. 56C. PVRIG+TIGIT+PD1+ CELLS ARE THE HIGHEST % AND MOSTEXHAUSTED OF CD8+ TILS. PVRIG+TIGIT+PD1+ CD8+ TILs are highly prevalentand have an exhausted phenotype. A) CD8⁺ TILs from human cancers werestained for PD-1, PVRIG, and TIGIT. The percentage of CD8⁺ TILs thatexpress combinations of PD-1, PVRIG, or TIGIT on CD8⁺ T cells wasdetermined by Boolean gating. Each dot represents an individual tumorsample. B) CD8⁺ TILs from human cancers were stained for cell surfacePD1, PVRIG, and TIGIT, permeabilized, and stained for intracellularEomes and T-bet. The percentage of Eomes+T-bet- CD8⁺ T cells are shown.A paired Student's t-test was performed and p-values shown. C) Thepercentage of Eomes⁺T-bet- CD8⁺ T cells expressing PD-1, PVRIG, andTIGIT was determined across multiple human cancers.

FIG. 57 . RELATIVE EXPRESSION OF PVRL2 VERSUS PVR VARIES BY TUMOR TYPE.Relative RNA and protein expression of PVRL2 and PVR across differenthuman tumors. RNA expression of PVRL2 and PVR from the TCGA was plottedas a ratio of PVRL2 relative to PVR across multiple human tumors (lefthand panel). Tumors with higher PVRL2 RNA expression compared to PVRinclude breast, ovarian, prostate, endometrial, bladder, pancreatic andlung. The ratio of protein expression (gMFI) of PVRL2 relative to PVR onCD45- tumor cells is plotted from dissociated human tumors (right handpanel). Each dot represents an individual tumor sample. Tumors withhigher PVRL2 protein expression compared to PVR include ovary, breast,endometrial, lung, prostate, oral cavity and stomach. Higher RNAexpression correlates with higher protein levels for PVRL2 acrossseveral tumors, including breast, ovarian, endometrial, prostate, andlung cancers.

FIG. 58A-FIG. 58B. PVRL2+PVR- TUMOR CELLS AND APCs EXIST IN HUMANTUMORS. PVRL2+PVR- tumor cells and APCs are present in human tumors.PVRL2 and PVR expression from dissociated tumors determined by FACS onA) CD45-tumor cells, and B) cDC2 (CD1c⁺CD14-HLA-DR^(hi)Lin⁻CD141⁻) andCD14⁺ TAMs is plotted. PVRL2+PVR- tumor cells and APCs are representedas red dots in the percent positive plots. Representative FACS plots forPVRL2 and PVR expression (blue) as compared to an IgG isotype control(red) are shown for ovarian and endometrial tumors.

FIG. 59A-FIG. 59B. CHA7.518.1.H4(S241P) +CPA.9.083.H4(S241P) COMBO HASACTIVITY PEMBROLIZUMAB ON PRIMARY CD3+ TILS. CHA7.518.1.H4(S241P) and/orCPA.9.083.H4(S241P) have similar or greater potency than Pembrolizumabon freshly isolated human TILs. A) Human tumors obtained within 24 hoursof surgical resection were dissociated and CD3+ TILs were purified.Isolated CD3+ TILs were co-cultured with a modified Mel-624 tumor cellline, expressing surface bound anti-CD3, and the indicated antibodies at10 μg/ml. B) IFN-γ secretion in the conditioned media was measured at 72hours. The percentage change in IFN-γ for each treatment over the hIgGisotype control is shown.

FIG. 60A-FIG. 60C. BLOCKADE OF PVRIG/PVRL2 INDUCES PD-1 AND TIGITEXPRESSION. Blockade of PVRIG/PVRL2 induces PD-1 & TIGIT expression.CMVpp65⁻specific CD8⁺ T cells from 2 donors were co-cultured withPanc.05.04, CMVpp65 peptide, and the indicated antibodies at 10 μg/mlfor 18 hrs. Cells were stained and the percentage of A) TIGIT⁺, B)PD-1⁺, and C) LAG3⁺ CD8⁺ T cells following each treatment is shown.

FIG. 61A-FIG. 61C. TRIPLE COMBINATION SHOWED IMPROVED ANTITUMOREFFICACY. A) Growth kinetics of CT26 tumors in a minimal disease model.Groups of 10 female Balb/c were inoculated with CT26 cells in the rightflank. I.p. antibody administration began when tumors reached a desiredmean volume (30-60 mm³). Mice were treated with anti-TIGIT mIgG1 oranti-PVRIG mIgG1 at 10 mg/kg, anti-PD-L1 mIgG1 at 3 mg/kg, and controlisotype at 10 mg/kg either as dual or triple combination, 3 timesbiweekly for a total of 6 doses. TGI with anti-TIGIT mIgG1 incombination was calculated by % TGI=[1-(average tumor volume of testarticle divided by average tumor volume of control article) *100]. Theasterisk (***, ****) indicate p<0.001 or p<0.0001, respectively, fordifferences between dual or triple combination over isotype control,versus dual or triple combination group by 2-way ANOVA. B) Spider plotsof individual tumor volumes of each mouse in the three treatment groupswere measured until tumor volumes of >1500 mm³ or 45 days (studyendpoints) were reached. C) Kaplan-Meier survival curves of mice treatedin the three different treatment groups. A log-rank (Mantel-Cox) testrevealed a p-value<0.0001, a 90% survival in mice treated tripleantibody combination versus 40% survival in mice in dual antibodycombination treatment.

FIG. 62A-FIG. 62I depicts the sequences of exemplary anti-PD-L1antibodies.

FIG. 63A-FIG. 63AAAA depicts the sequences of numerous exemplary PVRIGantibodies.

IV. DETAILED DESCRIPTION OF THE INVENTION

A. Introduction

Therapeutic antibodies directed against immune checkpoint inhibitorssuch as PD-1 are showing great promise in limited circumstances in theclinic for the treatment of cancer. Cancer can be considered as aninability of the patient to recognize and eliminate cancerous cells. Inmany instances, these transformed (e.g. cancerous) cells counteractimmunosurveillance. There are natural control mechanisms that limitT-cell activation in the body to prevent unrestrained T-cell activity,which can be exploited by cancerous cells to evade or suppress theimmune response. Restoring the capacity of immune effectorcells-especially T cells-to recognize and eliminate cancer is the goalof immunotherapy. The field of immuno-oncology, sometimes referred to as“immunotherapy” is rapidly evolving, with several recent approvals of Tcell checkpoint inhibitory antibodies such as Yervoy®, Keytruda® andOpdivo®. These antibodies are generally referred to as “checkpointinhibitors” because they block normally negative regulators of T cellimmunity. It is generally understood that a variety of immunomodulatorysignals, both costimulatory and coinhibitory, can be used to orchestratean optimal antigen-specific immune response.

Generally, these monoclonal antibodies bind to checkpoint inhibitorproteins such as CTLA-4 and PD-1, which under normal circumstancesprevent or suppress activation of cytotoxic T cells (CTLs). Byinhibiting the checkpoint protein, for example through the use ofantibodies that bind these proteins, an increased T cell responseagainst tumors can be achieved. That is, these cancer checkpointproteins suppress the immune response; when the proteins are blocked,for example using antibodies to the checkpoint protein, the immunesystem is activated, leading to immune stimulation, resulting intreatment of conditions such as cancer and infectious disease.

The present invention is directed to compositions and methods of usingseveral anti-checkpoint inhibitors in combination, so as to result inbetter patient outcomes. In particular, combinations of anti-TIGIT,anti-PVRIG and anti-PD-1 antibodies are contemplated. Furthermore, thesemethods are particularly useful in combination with an evaluation ofPD-L1 expression levels from the patient tumor. If the percentage ofPD-L1 positive tumor cells or immune cells is greater than 1% (>1%)compared to the same tumor cells stained with antibody relevant isotypecontrol antibody for the antibodies used then a triple combination ofanti-TIGIT, anti-PVRIG and anti-PD-1 antibodies should be administered.Whereas, patients with a frequency of PD-L1 positive tumor cells orimmune cells below 1% (<1%) compared to the isotype control should beadministered a double combination of anti-TIGIT and anti-PVRIGantibodies.

As discussed herein, TIGIT is a co-inhibitory receptor that is highlyexpressed on effector & regulatory (Treg) CD4+ T cells, effector CD8+ Tcells, and NK cells. TIGIT has been shown to attenuate immune responseby (1) direct signaling, (2) inducing ligand signaling, and (3)competition with and disruption of signaling by the costimulatoryreceptor CD226 (also known as DNAM-1).

Human Poliovirus Receptor Related Immunoglobulin Domain ContainingProtein, or “PVRIG”, is expressed on the cell surface of NK and T-cellsand shares several similarities to other known immune checkpoints. PVRIGhas been validated as a checkpoint inhibitor, see U.S. Ser. Nos.62/118,208, 62/141,120, 62/235,823, 62/376,334, 15/048,967, 62/376,335,62/417,217 and 62/477,974, all of which are expressly incorporatedherein by reference in their entirety and in particular for thesequences of the antibodies, figures and figure legends therein. Asshown in those documents, PVRL2 was identified/confirmed to be thecounterpart of PVRIG. Antibodies that bind to PVRIG were generated, andthen a subset of those were identified that both bind to PVRIG and blockthe interaction of PVRIG and PVLR2. When PVRIG is bound by its ligand(PVRL2), an inhibitory signal is elicited which acts to attenuate theimmune response of NK and T-cells against a target cell (i.e. analogousto PD-1/PDL1). Blocking the binding of PVRL2 to PVRIG shuts-off thisinhibitory signal of PVRIG and as a result modulates the immune responseof NK and T-cells.

PD-1, or “programmed cell death protein 1”, is a known checkpointinhibitor. There are two approved anti-PD-1 antibodies, pembrolizumab(Keytruda®), cemiplimab (REGN2810), and nivolumab (Opdivo®) and manymore in development (including, but not limited to, pidilizumab, BAP049clones as listed in WO2015/112900 (the sequences of which are expresslyincorporated herein by reference), antibody 317-4B6 as listed inWO2015/035606 (the sequence of which is expressly incorporated herein byreference), antibody APE2058 as listed in US2016/0075783 (the sequenceof which is expressly incorporated herein by reference).

There are three approved anti-PD-L1 antibodies, atezolizumab(TECENTRIQ®), avelumab (BAVENCIO®), and durvalumab, as well as otheranti-PD-L1 antibodies in development.

Functional effects of the combinations of these antibodies on NK andT-cells can be assessed in vitro (and in some cases in vivo, asdescribed more fully below) by measuring changes in the followingparameters: proliferation, cytokine release and cell-surface makers.Accordingly, functional effects of the anti- TIGIT antibodies on NK,effector T, and Treg cells can be assessed in vitro (and in some cases,in vivo, as described more fully below) by measuring changes in thefollowing parameters: proliferation, cytokine release and cell-surfacereceptors. For NK cells, increases in cell proliferation, cytotoxicity(ability to kill target cells as measured by increases in CD107a,granzyme, and perforin expression, or by directly measuring target cellskilling), cytokine production (e.g. IFN-γ and TNF), and cell surfacereceptor expression (e.g. CD25) is indicative of immune modulation, e.g.enhanced killing of cancer cells. For effector T and Treg-cells,increases in proliferation, increases in expression of cell surfacereceptors of activation (e.g. CD25, CD69, CD137, and PD-1), cytotoxicity(ability to kill target cells, as mentioned above), and cytokineproduction (e.g. IL-2, IL-4, IL-6, IFN-γ, TNF-α, IL-10, IL-17A) areindicative of immune modulation, e.g. enhanced killing of cancer cells.Accordingly, assessment of treatment can be done using assays thatevaluate one or more of the following: (i) increases in immune response,(ii) increases in activation of γβ and/or γδ T cells, (iii) increases incytotoxic T cell activity, (iv) increases in NK and/or NKT cellactivity, (v) alleviation of αβ and/or γδ T-cell suppression, (vi)increases in pro-inflammatory cytokine secretion, (vii) increases inIL-2 secretion; (viii) increases in interferon-γ production, (ix)increases in Th1 response, (x) decreases in Th2 response, (xi) decreasesor eliminates cell number and/or activity of at least one of regulatoryT cells.

In particular, any one of the assays shown in Example 1 can be used tomeasure T cell activation and/or suppression of T cell inhibition.

Thus, in some embodiments the invention provides the use of combinationtherapies of anti-TIGIT, anti-PVRIG and anti-PD-1 antibodies (or justanti-TIGIT and anti-PVRIG antibodies in some cases as outlined herein)to perform one or more of the following in a subject in need thereof:(a) upregulating pro-inflammatory cytokines; (b) increasing T-cellproliferation and/or expansion; (c) increasing interferon- or TNF-aproduction by T-cells; (d) increasing IL-2 secretion; (e) stimulatingantibody responses; (f) inhibiting cancer cell growth; (g) promotingantigenic specific T cell immunity; (h) promoting CD4+ and/or CD8+ Tcell activation; (i) alleviating Treg-mediated-cell suppression; (j)promoting NK cell activity; (k) promoting apoptosis or lysis of cancercells; and/or (l) cytotoxic or cytostatic effect on cancer cells.

Accordingly, the present invention provides anti-TIGIT, anti-PVRIG andanti-PD-1 antibodies for use in combination therapies, and inconjunction with diagnostic assays measuring the levels of one or moreof TIGIT, PVRIG and PD-1 expression, and/or measuring the levels of theligands of TIGIT (e.g., PVR), PVRIG (PVRL2) and PD-1 (PD-L1).

B. Definitions

In order that the application may be more completely understood, severaldefinitions are set forth below. Such definitions are meant to encompassgrammatical equivalents.

By “ablation” herein is meant a decrease or removal of activity. In someembodiments, it is useful to remove activity from the constant domainsof the antibodies. Thus for example, “ablating FcγR binding” means theFc region amino acid variant has less than 50% starting binding ascompared to an Fc region not containing the specific variant, with lessthan 70-80-90-95-98% loss of activity being preferred, and in general,with the activity being below the level of detectable binding in aBiacore assay. As shown in FIG. 1 , one ablation variant in the IgG1constant region is the N297A variant, which removes the nativeglycosylation site and significantly reduces the FcγRIIIa binding andthus reduces the antibody dependent cell-mediated cytotoxicity (ADCC).

By “antigen binding domain” or “ABD” herein is meant a set of sixComplementary Determining Regions (CDRs) that, when present as part of apolypeptide sequence, specifically binds a target antigen as discussedherein. Thus, a “TIGIT antigen binding domain” binds TIGIT antigen (thesequence of which is shown in FIG. 2 ) as outlined herein. As is knownin the art, these CDRs are generally present as a first set of variableheavy CDRs (vhCDRs or VHCDRs) and a second set of variable light CDRs(vlCDRs or V_(L)CDRs), each comprising three CDRs: vhCDR1, vhCDR2,vhCDR3 for the heavy chain and vlCDR1, vlCDR2 and vlCDR3 for the light.The CDRs are present in the variable heavy and variable light domains,respectively, and together form an Fv region. Thus, in some cases, thesix CDRs of the antigen binding domain are contributed by a variableheavy and variable light chain. In a “Fab” format, the set of 6 CDRs arecontributed by two different polypeptide sequences, the variable heavydomain (vh or V_(H); containing the vhCDR1, vhCDR2 and vhCDR3) and thevariable light domain (vl or V_(L); containing the vlCDR1, vlCDR2 andvlCDR3), with the C-terminus of the vh domain being attached to theN-terminus of the CH1 domain of the heavy chain and the C-terminus ofthe vl domain being attached to the N-terminus of the constant lightdomain (and thus forming the light chain).

By “modification” herein is meant an amino acid substitution, insertion,and/or deletion in a polypeptide sequence or an alteration to a moietychemically linked to a protein. For example, a modification may be analtered carbohydrate or PEG structure attached to a protein. By “aminoacid modification” herein is meant an amino acid substitution,insertion, and/or deletion in a polypeptide sequence. For clarity,unless otherwise noted, the amino acid modification is always to anamino acid coded for by DNA, e.g. the 20 amino acids that have codons inDNA and RNA.

By “amino acid substitution” or “substitution” herein is meant thereplacement of an amino acid at a particular position in a parentpolypeptide sequence with a different amino acid. In particular, in someembodiments, the substitution is to an amino acid that is not naturallyoccurring at the particular position, either not naturally occurringwithin the organism or in any organism. For example, the substitutionN297A refers to a variant polypeptide, in this case an Fc variant, inwhich the asparagine at position 297 is replaced with alanine. Forclarity, a protein which has been engineered to change the nucleic acidcoding sequence but not change the starting amino acid (for exampleexchanging CGG (encoding arginine) to CGA (still encoding arginine) toincrease host organism expression levels) is not an “amino acidsubstitution”; that is, despite the creation of a new gene encoding thesame protein, if the protein has the same amino acid at the particularposition that it started with, it is not an amino acid substitution.

By “amino acid insertion” or “insertion” as used herein is meant theaddition of an amino acid sequence at a particular position in a parentpolypeptide sequence. For example, −233E or 233E designates an insertionof glutamic acid after position 233 and before position 234.Additionally, −233ADE or A233ADE designates an insertion of AlaAspGluafter position 233 and before position 234.

By “amino acid deletion” or “deletion” as used herein is meant theremoval of an amino acid sequence at a particular position in a parentpolypeptide sequence. For example, E233− or E233#, E233( ) or E233deldesignates a deletion of glutamic acid at position 233. Additionally,EDA233− or EDA233# designates a deletion of the sequence GluAspAla thatbegins at position 233.

By “variant protein” or “protein variant”, or “variant” as used hereinis meant a protein that differs from that of a parent protein by virtueof at least one amino acid modification. Protein variant may refer tothe protein itself, a composition comprising the protein, or the aminosequence that encodes it. Preferably, the protein variant has at leastone amino acid modification compared to the parent protein, e.g. fromabout one to about seventy amino acid modifications, and preferably fromabout one to about five amino acid modifications compared to the parent.As described below, in some embodiments the parent polypeptide, forexample an Fc parent polypeptide, is a human wild type sequence, such asthe Fc region from IgG1, IgG2, IgG3 or IgG4, although human sequenceswith variants can also serve as “parent polypeptides”. The proteinvariant sequence herein will preferably possess at least about 80%identity with a parent protein sequence, and most preferably at leastabout 90% identity, more preferably at least about 95-98-99% identity.Variant protein can refer to the variant protein itself, compositionscomprising the protein variant, or the DNA sequence that encodes it.Accordingly, by “antibody variant” or “variant antibody” as used hereinis meant an antibody that differs from a parent antibody by virtue of atleast one amino acid modification, “IgG variant” or “variant IgG” asused herein is meant an antibody that differs from a parent IgG (again,in many cases, from a human IgG sequence) by virtue of at least oneamino acid modification, and “immunoglobulin variant” or “variantimmunoglobulin” as used herein is meant an immunoglobulin sequence thatdiffers from that of a parent immunoglobulin sequence by virtue of atleast one amino acid modification. “Fc variant” or “variant Fc” as usedherein is meant a protein comprising an amino acid modification in an Fcdomain. The Fc variants of the present invention are defined accordingto the amino acid modifications that compose them. Thus, for example,S241P or S228P is a hinge variant with the substitution proline atposition 228 relative to the parent IgG4 hinge polypeptide, wherein thenumbering S228P is according to the EU index and the S241P is the Kabatnumbering. The EU index or EU index as in Kabat or EU numbering schemerefers to the numbering of the EU antibody (Edelman et al., 1969, ProcNatl Acad Sci USA 63:78-85, hereby entirely incorporated by reference.)The modification can be an addition, deletion, or substitution.Substitutions can include naturally occurring amino acids and, in somecases, synthetic amino acids. Examples include U.S. Pat. No. 6,586,207;WO 98/48032; WO 03/073238; US2004-0214988A1; WO 05/35727A2; WO05/74524A2; J. W. Chin et al., (2002), Journal of the American ChemicalSociety 124:9026-9027; J. W. Chin, & P. G. Schultz, (2002), ChemBioChem11:1135-1137; J. W. Chin, et al., (2002), PICAS United States of America99:11020-11024; and, L. Wang, & P. G. Schultz, (2002), Chem. 1-10, allentirely incorporated by reference.

As used herein, “protein” herein is meant at least two covalentlyattached amino acids, which includes proteins, polypeptides,oligopeptides and peptides. The peptidyl group may comprise naturallyoccurring amino acids and peptide bonds, or synthetic peptidomimeticstructures, i.e. “analogs”, such as peptoids (see Simon et al., PNAS USA89(20):9367 (1992), entirely incorporated by reference). The amino acidsmay either be naturally occurring or synthetic (e.g. not an amino acidthat is coded for by DNA); as will be appreciated by those in the art.For example, homo-phenylalanine, citrulline, ornithine and noreleucineare considered synthetic amino acids for the purposes of the invention,and both D- and L-(R or S) configured amino acids may be utilized. Thevariants of the present invention may comprise modifications thatinclude the use of synthetic amino acids incorporated using, forexample, the technologies developed by Schultz and colleagues, includingbut not limited to methods described by Cropp & Shultz, 2004, TrendsGenet. 20(12):625-30, Anderson et al., 2004, Proc Natl Acad Sci USA 101(2):7566-71, Zhang et al., 2003, 303(5656):371-3, and Chin et al., 2003,Science 301(5635):964-7, all entirely incorporated by reference. Inaddition, polypeptides may include synthetic derivatization of one ormore side chains or termini, glycosylation, PEGylation, circularpermutation, cyclization, linkers to other molecules, fusion to proteinsor protein domains, and addition of peptide tags or labels.

By “residue” as used herein is meant a position in a protein and itsassociated amino acid identity. For example, Asparagine 297 (alsoreferred to as Asn297 or N297) is a residue at position 297 in the humanantibody IgG1.

By “Fab” or “Fab region” as used herein is meant the polypeptide thatcomprises the VH, CH1, VL, and CL immunoglobulin domains. Fab may referto this region in isolation, or this region in the context of a fulllength antibody or antibody fragment.

By “Fv” or “Fv fragment” or “Fv region” as used herein is meant apolypeptide that comprises the VL and VH domains of a single antibody.As will be appreciated by those in the art, these generally are made upof two chains.

By “single chain Fv” or “scFv” herein is meant a variable heavy domaincovalently attached to a variable light domain, generally using a scFvlinker as discussed herein, to form a scFv or scFv domain. A scFv domaincan be in either orientation from N- to C-terminus (vh-linker-vl orvl-linker-vh). In general, the linker is a scFv linker as is generallyknown in the art, with the linker peptide predominantly including thefollowing amino acid residues: Gly, Ser, Ala, or Thr. The linker peptideshould have a length that is adequate to link two molecules in such away that they assume the correct conformation relative to one another sothat they retain the desired activity. In one embodiment, the linker isfrom about 1 to 50 amino acids in length, preferably about 1 to 30 aminoacids in length. In one embodiment, linkers of 1 to 20 amino acids inlength may be used, with from about 5 to about 10 amino acids findinguse in some embodiments. Useful linkers include glycine-serine polymers,including for example (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n isan integer of at least one (and generally from 3 to 4), glycine-alaninepolymers, alanine-serine polymers, and other flexible linkers.Alternatively, a variety of nonproteinaceous polymers, including but notlimited to polyethylene glycol (PEG), polypropylene glycol,polyoxyalkylenes, or copolymers of polyethylene glycol and polypropyleneglycol, may find use as linkers, that is may find use as linkers.

By “IgG subclass modification” or “isotype modification” as used hereinis meant an amino acid modification that converts one amino acid of oneIgG isotype to the corresponding amino acid in a different, aligned IgGisotype. For example, because IgG1 comprises a tyrosine and IgG2 aphenylalanine at EU position 296, a F296Y substitution in IgG2 isconsidered an IgG subclass modification. Similarly, because IgG1 has aproline at position 241 and IgG4 has a serine there, an IgG4 moleculewith a S241P is considered an IgG subclass modification. Note thatsubclass modifications are considered amino acid substitutions herein.

By “non-naturally occurring modification” as used herein is meant anamino acid modification that is not isotypic. For example, because noneof the IgGs comprise an asparagine at position 297, the substitutionN297A in IgG1, IgG2, IgG3, or IgG4 (or hybrids thereof) is considered anon-naturally occurring modification.

By “amino acid” and “amino acid identity” as used herein is meant one ofthe 20 naturally occurring amino acids that are coded for by DNA andRNA.

By “effector function” as used herein is meant a biochemical event thatresults from the interaction of an antibody Fc region with an Fcreceptor or ligand. Effector functions include but are not limited toADCC, ADCP, and CDC. In many cases, it is desirable to ablate most orall effector functions using either different IgG isotypes (e.g. IgG4)or amino acid substitutions in the Fc domain; however, preservingbinding to the FcRn receptor is desirable, as this contributes to thehalf-life of the antibodies in human serum.

By “IgG Fc ligand” as used herein is meant a molecule, preferably apolypeptide, from any organism that binds to the Fc region of an IgGantibody to form an Fc/Fc ligand complex. Fc ligands include but are notlimited to FcγRIs, FcγRIIs, FcγRIIIs, FcRn, C1q, C3, mannan bindinglectin, mannose receptor, staphylococcal protein A, streptococcalprotein G, and viral FcγR. Fc ligands also include Fc receptor homologs(FcRH), which are a family of Fc receptors that are homologous to theFcγRs (Davis et al., 2002, Immunological Reviews 190:123-136, entirelyincorporated by reference). Fc ligands may include undiscoveredmolecules that bind Fc. Particular IgG Fc ligands are FcRn and Fc gammareceptors. By “Fc ligand” as used herein is meant a molecule, preferablya polypeptide, from any organism that binds to the Fc region of anantibody to form an Fc/Fc ligand complex.

By “parent polypeptide” as used herein is meant a starting polypeptidethat is subsequently modified to generate a variant. The parentpolypeptide may be a naturally occurring polypeptide, or a variant orengineered version of a naturally occurring polypeptide. Parentpolypeptide may refer to the polypeptide itself, compositions thatcomprise the parent polypeptide, or the amino acid sequence that encodesit. Accordingly, by “parent immunoglobulin” as used herein is meant anunmodified immunoglobulin polypeptide that is modified to generate avariant, and by “parent antibody” as used herein is meant an unmodifiedantibody that is modified to generate a variant antibody. It should benoted that “parent antibody” includes known commercial, recombinantlyproduced antibodies as outlined below.

By “Fc” or “Fc region” or “Fc domain” as used herein is meant thepolypeptide comprising the constant region of an antibody excluding thefirst constant region immunoglobulin domain and in some cases, part ofthe hinge. Thus Fc refers to the last two constant region immunoglobulindomains of IgA, IgD, and IgG, the last three constant regionimmunoglobulin domains of IgE and IgM, and the flexible hinge N-terminalto these domains. For IgA and IgM, Fc may include the J chain. For IgG,the Fc domain comprises immunoglobulin domains Cγ2 and Cγ3 (Cγ2 and Cγ3)and the lower hinge region between Cγ1 (Cγ1) and Cγ2 (Cγ2). Although theboundaries of the Fc region may vary, the human IgG heavy chain Fcregion is usually defined to include residues C226 or P230 to itscarboxyl-terminus, wherein the numbering is according to the EU index asin Kabat. In some embodiments, as is more fully described below, aminoacid modifications are made to the Fc region, for example to alterbinding to one or more FcγR receptors or to the FcRn receptor.

By “heavy constant region” herein is meant the CH1-hinge-CH2-CH3 portionof an antibody.

By “position” as used herein is meant a location in the sequence of aprotein. Positions may be numbered sequentially, or according to anestablished format, for example the EU index for antibody numbering.

By “target antigen” as used herein is meant the molecule that is boundspecifically by the variable region of a given antibody. The targetantigen of interest herein is TIGIT, usually human TIGIT and optionallycyno TIGIT, the sequences of which are shown in.

By “target cell” as used herein is meant a cell that expresses a targetantigen.

By “variable region” as used herein is meant the region of animmunoglobulin that comprises one or more Ig domains substantiallyencoded by any of the Vκ (V.kappa), Vλ (V.lamda), and/or VH genes thatmake up the kappa, lambda, and heavy chain immunoglobulin genetic locirespectively.

By “wild type or WT” herein is meant an amino acid sequence or anucleotide sequence that is found in nature, including allelicvariations. A WT protein has an amino acid sequence or a nucleotidesequence that has not been intentionally modified.

The antibodies of the present invention are generally isolated orrecombinant. “Isolated,” when used to describe the various polypeptidesdisclosed herein, means a polypeptide that has been identified andseparated and/or recovered from a cell or cell culture from which it wasexpressed. Ordinarily, an isolated polypeptide will be prepared by atleast one purification step. An “isolated antibody,” refers to anantibody which is substantially free of other antibodies havingdifferent antigenic specificities. “Recombinant” means the antibodiesare generated using recombinant nucleic acid techniques in exogeneoushost cells.

“Specific binding” or “specifically binds to” or is “specific for” aparticular antigen or an epitope means binding that is measurablydifferent from a non-specific interaction. Specific binding can bemeasured, for example, by determining binding of a molecule compared tobinding of a control molecule, which generally is a molecule of similarstructure that does not have binding activity. For example, specificbinding can be determined by competition with a control molecule that issimilar to the target.

Specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KD for an antigen orepitope of at least about 10⁻⁹ M, at least about 10⁻¹⁰ M, at least about10⁻¹¹ M, at least about 10⁻¹² M, at least about 10⁻¹³ M, at least about10⁻¹⁴ M, at least about 10⁻¹⁵ M, where KD refers to a dissociation rateof a particular antibody-antigen interaction. Typically, an antibodythat specifically binds an antigen will have a KD that is 20-, 50-,100-, 500-, 1000-, 5,000-, 10,000- or more times greater for a controlmolecule relative to the antigen or epitope.

Also, specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KA or Ka for an antigenor epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- ormore times greater for the epitope relative to a control, where KA or Karefers to an association rate of a particular antibody-antigeninteraction. Binding affinity is generally measured using surfaceplasmon resonance (e.g. Biacore assay) and flow cytometry withantigen-expressing cells.

V. ANTIBODIES

As is discussed below, the term “antibody” is used generally.Traditional antibody structural units typically comprise a tetramer.Each tetramer is typically composed of two identical pairs ofpolypeptide chains, each pair having one “light” (typically having amolecular weight of about 25 kDa) and one “heavy” chain (typicallyhaving a molecular weight of about 50-70 kDa). Human light chains areclassified as kappa and lambda light chains. The present invention isdirected to antibodies that generally are based on the IgG class, whichhas several subclasses, including, but not limited to IgG1, IgG2, IgG3,and IgG4. In general, IgG1, IgG2 and IgG4 are used more frequently thanIgG3. It should be noted that IgG1 has different allotypes withpolymorphisms at 356 (D or E) and 358 (L or M). The sequences depictedherein use the 356D/358M allotype, however the other allotype isincluded herein. That is, any sequence inclusive of an IgG1 Fc domainincluded herein can have 356E/358L replacing the 356D/358M allotype.

The amino-terminal portion of each chain includes a variable region ofabout 100 to 110 or more amino acids primarily responsible for antigenrecognition, generally referred to in the art and herein as the “Fvdomain” or “Fv region”. In the variable region, three loops are gatheredfor each of the V domains of the heavy chain and light chain to form anantigen-binding site. Each of the loops is referred to as acomplementarity-determining region (hereinafter referred to as a “CDR”),in which the variation in the amino acid sequence is most significant.“Variable” refers to the fact that certain segments of the variableregion differ extensively in sequence among antibodies. Variabilitywithin the variable region is not evenly distributed. Instead, the Vregions consist of relatively invariant stretches called frameworkregions (FRs) of 15-30 amino acids separated by shorter regions ofextreme variability called “hypervariable regions” that are each 9-15amino acids long or longer.

Each VH and VL is composed of three hypervariable regions(“complementary determining regions,” “CDRs”) and four FRs, arrangedfrom amino-terminus to carboxy-terminus in the following order:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

The hypervariable region generally encompasses amino acid residues fromabout amino acid residues 24-34 (LCDR1; “L” denotes light chain), 50-56(LCDR2) and 89-97 (LCDR3) in the light chain variable region and aroundabout 31-35B (HCDR1; “H” denotes heavy chain), 50-65 (HCDR2), and 95-102(HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OFPROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991) and/or thoseresidues forming a hypervariable loop (e.g. residues 26-32 (LCDR1),50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chainvariable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917.Specific CDRs of the invention are described below.

As will be appreciated by those in the art, the exact numbering andplacement of the CDRs can be different among different numberingsystems. However, it should be understood that the disclosure of avariable heavy and/or variable light sequence includes the disclosure ofthe associated (inherent) CDRs. Accordingly, the disclosure of eachvariable heavy region is a disclosure of the vhCDRs (e.g. vhCDR1, vhCDR2and vhCDR3) and the disclosure of each variable light region is adisclosure of the vhCDRs (e.g. vlCDR1, vlCDR2 and vlCDR3). A usefulcomparison of CDR numbering is as below, see Lafranc et al., Dev. Comp.Immunol. 27(1):55-77 (2003):

Kabat + Clothia IMGT Kabat AbM Chothia Contact vhCDR1 26-35 27-38 31-3526-35 26-32 30-35 vhCDR2 50-65 56-65 50-65 50-58 53-55 47-58 vhCDR3 95-102 105-117  95-102  95-102  96-101  93-101 vlCDR1 24-34 27-38 24-3424-34 26-32 30-36 vlCDR2 50-56 56-65 50-56 50-56 50-52 46-55 vlCDR389-97 105-117 89-97 89-97 91-96 89-96

Throughout the present specification, the Kabat numbering system isgenerally used when referring to a residue in the variable domain(approximately, residues 1-107 of the light chain variable region andresidues 1-113 of the heavy chain variable region) and the hinge and theEU numbering system for Fc regions (e.g, Kabat et al., supra (1991)).

The present invention provides a large number of different CDR sets. Inthis case, a “full CDR set” comprises the three variable light and threevariable heavy CDRs, e.g. a vlCDR1, vlCDR2, vlCDR3, vhCDR1, vhCDR2 andvhCDR3. These can be part of a larger variable light or variable heavydomain, respectfully. In addition, as more fully outlined herein, thevariable heavy and variable light domains can be on separate polypeptidechains, when a heavy and light chain is used, or on a single polypeptidechain in the case of scFv sequences.

The CDRs contribute to the formation of the antigen-binding, or morespecifically, epitope binding site of antibodies. “Epitope” refers to adeterminant that interacts with a specific antigen binding site in thevariable region of an antibody molecule known as a paratope. Epitopesare groupings of molecules such as amino acids or sugar side chains andusually have specific structural characteristics, as well as specificcharge characteristics. A single antigen may have more than one epitope.

The epitope may comprise amino acid residues directly involved in thebinding (also called immunodominant component of the epitope) and otheramino acid residues, which are not directly involved in the binding,such as amino acid residues which are effectively blocked by thespecifically antigen binding peptide; in other words, the amino acidresidue is within the footprint of the specifically antigen bindingpeptide.

Epitopes may be either conformational or linear. A conformationalepitope is produced by spatially juxtaposed amino acids from differentsegments of the linear polypeptide chain. A linear epitope is oneproduced by adjacent amino acid residues in a polypeptide chain.Conformational and nonconformational epitopes may be distinguished inthat the binding to the former but not the latter is lost in thepresence of denaturing solvents.

An epitope typically includes at least 3, and more usually, at least 5or 8-10 amino acids in a unique spatial conformation. Antibodies thatrecognize the same epitope can be verified in a simple immunoassayshowing the ability of one antibody to block the binding of anotherantibody to a target antigen, for example “binning.” As outlined below,the invention not only includes the enumerated antigen binding domainsand antibodies herein, but those that compete for binding with theepitopes bound by the enumerated antigen binding domains.

The carboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function. Kabat et al. collectednumerous primary sequences of the variable regions of heavy chains andlight chains. Based on the degree of conservation of the sequences, theyclassified individual primary sequences into the CDR and the frameworkand made a list thereof (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5thedition, NIH publication, No. 91-3242, E. A. Kabat et al., entirelyincorporated by reference).

In the IgG subclass of immunoglobulins, there are several immunoglobulindomains in the heavy chain. By “immunoglobulin (Ig) domain” herein ismeant a region of an immunoglobulin having a distinct tertiarystructure. Of interest in the present invention are the heavy chaindomains, including, the constant heavy (CH) domains and the hingedomains. In the context of IgG antibodies, the IgG isotypes each havethree CH regions. Accordingly, “CH” domains in the context of IgG are asfollows: “CH1” refers to positions 118-220 according to the EU index asin Kabat. “CH2” refers to positions 237-340 according to the EU index asin Kabat, and “CH3” refers to positions 341-447 according to the EUindex as in Kabat. As shown herein and described below, the pI variantscan be in one or more of the CH regions, as well as the hinge region,discussed below.

Another type of Ig domain of the heavy chain is the hinge region. By“hinge” or “hinge region” or “antibody hinge region” or “immunoglobulinhinge region” herein is meant the flexible polypeptide comprising theamino acids between the first and second constant domains of anantibody. Structurally, the IgG CH1 domain ends at EU position 220, andthe IgG CH2 domain begins at residue EU position 237. Thus for IgG theantibody hinge is herein defined to include positions 221 (D221 in IgG1)to 236 (G236 in IgG1), wherein the numbering is according to the EUindex as in Kabat. In some embodiments, for example in the context of anFc region, the lower hinge is included, with the “lower hinge” generallyreferring to positions 226 or 230.

The light chain generally comprises two domains, the variable lightdomain (containing the light chain CDRs and together with the variableheavy domains forming the Fv region), and a constant light chain region(often referred to as CL or C_(κ)).

Another region of interest for additional substitutions, outlined below,is the Fc region.

A. Chimeric and Humanized Antibodies

In some embodiments, the antibodies herein can be derived from a mixturefrom different species, e.g. a chimeric antibody and/or a humanizedantibody. In general, both “chimeric antibodies” and “humanizedantibodies” refer to antibodies that combine regions from more than onespecies. For example, “chimeric antibodies” traditionally comprisevariable region(s) from a mouse (or rat, in some cases) and the constantregion(s) from a human. “Humanized antibodies” generally refer tonon-human antibodies that have had the variable-domain framework regionsswapped for sequences found in human antibodies. Generally, in ahumanized antibody, the entire antibody, except the CDRs, is encoded bya polynucleotide of human origin or is identical to such an antibodyexcept within its CDRs. The CDRs, some or all of which are encoded bynucleic acids originating in a non-human organism, are grafted into thebeta-sheet framework of a human antibody variable region to create anantibody, the specificity of which is determined by the engrafted CDRs.The creation of such antibodies is described in, e.g., WO 92/11018,Jones, 1986, Nature 321:522-525, Verhoeyen et al., 1988, Science239:1534-1536, all entirely incorporated by reference. “Backmutation” ofselected acceptor framework residues to the corresponding donor residuesis often required to regain affinity that is lost in the initial graftedconstruct (U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762;6,180,370; 5,859,205; 5,821,337; 6,054,297; 6,407,213, all entirelyincorporated by reference). The humanized antibody optimally also willcomprise at least a portion of an immunoglobulin constant region,typically that of a human immunoglobulin, and thus will typicallycomprise a human Fc region. Humanized antibodies can also be generatedusing mice with a genetically engineered immune system. Roque et al.,2004, Biotechnol. Prog. 20:639-654, entirely incorporated by reference.A variety of techniques and methods for humanizing and reshapingnon-human antibodies are well known in the art (See Tsurushita &Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biologyof B Cells, 533-545, Elsevier Science (USA), and references citedtherein, all entirely incorporated by reference). Humanization methodsinclude but are not limited to methods described in Jones et al., 1986,Nature 321:522-525; Riechmann et al.,1988; Nature 332:323-329; Verhoeyenet al., 1988, Science, 239:1534-1536; Queen et al., 1989, Proc Natl AcadSci, USA 86:10029-33; He et al., 1998, J. Immunol. 160: 1029-1035;Carter et al., 1992, Proc Natl Acad Sci USA 89:4285-9, Presta et al.,1997, Cancer Res. 57(20):4593-9; Gorman et al., 1991, Proc. Natl. Acad.Sci. USA 88:4181-4185; O′Connor et al., 1998, Protein Eng 11:321-8, allentirely incorporated by reference. Humanization or other methods ofreducing the immunogenicity of nonhuman antibody variable regions mayinclude resurfacing methods, as described for example in Roguska et al.,1994, Proc. Natl. Acad. Sci. USA 91:969-973, entirely incorporated byreference.

In certain embodiments, the antibodies of the invention comprise a heavychain variable region from a particular germline heavy chainimmunoglobulin gene and/or a light chain variable region from aparticular germline light chain immunoglobulin gene (with optionalmutations as is generally described herein). For example, suchantibodies may comprise or consist of a human antibody comprising heavyor light chain variable regions that are “the product of” or “derivedfrom” a particular germline sequence. A human antibody that is “theproduct of” or “derived from” a human germline immunoglobulin sequencecan be identified as such by comparing the amino acid sequence of thehuman antibody to the amino acid sequences of human germlineimmunoglobulins and selecting the human germline immunoglobulin sequencethat is closest in sequence (i.e., greatest % identity) to the sequenceof the human antibody. A human antibody that is “the product of” or“derived from” a particular human germline immunoglobulin sequence maycontain amino acid differences as compared to the germline sequence, dueto, for example, naturally-occurring somatic mutations or intentionalintroduction of site-directed mutation. However, a humanized antibodytypically is at least 90% identical in amino acids sequence to an aminoacid sequence encoded by a human germline immunoglobulin gene andcontains amino acid residues that identify the antibody as being derivedfrom human sequences when compared to the germline immunoglobulin aminoacid sequences of other species (e.g., murine germline sequences). Incertain cases, a humanized antibody may be at least 95, 96, 97, 98 or99%, or even at least 96%, 97%, 98%, or 99% identical in amino acidsequence to the amino acid sequence encoded by the germlineimmunoglobulin gene. Typically, a humanized antibody derived from aparticular human germline sequence will display no more than 10-20 aminoacid differences from the amino acid sequence encoded by the humangermline immunoglobulin gene. In certain cases, the humanized antibodymay display no more than 5, or even no more than 4, 3, 2, or 1 aminoacid difference from the amino acid sequence encoded by the germlineimmunoglobulin gene (again, prior to the introduction of any variantsherein; that is, the number of variants is generally low, prior to theintroduction of the variants of the invention).

In one embodiment, the parent antibody has been affinity matured, as isknown in the art. Structure-based methods may be employed forhumanization and affinity maturation, for example as described in U.S.Ser. No. 11/004,590. Selection based methods may be employed to humanizeand/or affinity mature antibody variable regions, including but notlimited to methods described in Wu et al., 1999, J. Mol. Biol.294:151-162; Baca et al., 1997, J. Biol. Chem. 272(16):10678-10684;Rosok et al., 1996, J. Biol. Chem. 271(37): 22611-22618; Rader et al.,1998, Proc. Natl. Acad. Sci. USA 95: 8910-8915; Krauss et al., 2003,Protein Engineering 16(10):753-759, all entirely incorporated byreference. Other humanization methods may involve the grafting of onlyparts of the CDRs, including but not limited to methods described inUSSN 09/810,510; Tan et al., 2002, J. Immunol. 169:1119-1125; DePascalis et al., 2002, J. Immunol. 169:3076-3084, all entirelyincorporated by reference.

B. Specific Anti-TIGIT Antibodies

The invention provides antigen binding domains, including full lengthantibodies, which contain a number of specific, enumerated sets of 6CDRs and defined variable heavy (vh, VH or V_(H)) and variable light(vl, VL or V_(L)), that bind to TIGIT.

In one embodiment, the anti-TIGIT antibody is an antibody comprising aset of six CDRs (vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2 and vlCDR3) fromCPA.9.083.H4(S241P) as depicted in FIG. 3 . In one embodiment, theanti-TIGIT antibody is an antibody comprising the variable heavy (vh)and variable light (vl) domains from CPA.9.083.H4(S241P) as depicted inFIG. 3 , linked to a human IgG constant domain of IgG1, IgG2, IgG3, IgG4and IgG4(S241P). In one embodiment, the anti-TIGIT antibody isCPA.9.083.H4(S241P).

In one embodiment, the anti-TIGIT antibody is an antibody comprising aset of six CDRs (vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2 and vlCDR3) fromCPA.9.086.H4(S241P) as depicted in FIG. 3 . In one embodiment, theanti-TIGIT antibody is an antibody comprising the variable heavy (vh)and variable light (vl) domains from CPA.9.086.H4(S241P) as depicted inFIG. 3 , linked to a human IgG constant domain of IgG1, IgG2, IgG3, IgG4and IgG4(S241P). In one embodiment, the anti-TIGIT antibody isCPA.9.086.H4(S241P).

In one embodiment, the anti-TIGIT antibody is an antibody comprising aset of six CDRs (vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2 and vlCDR3) fromCHA.9.547.7.H4(S241P) as depicted in FIG. 3 . In one embodiment, theanti-TIGIT antibody is an antibody comprising the variable heavy (vh)and variable light (vl) domains from CHA.9.547.7.H4(S241P) as depictedin FIG. 3 , linked to a human IgG constant domain of IgG1, IgG2, IgG3,IgG4 and IgG4(S241P). In one embodiment, the anti-TIGIT antibody isCHA.9.547.7.H4(S241P).

In one embodiment, the anti-TIGIT antibody is an antibody comprising aset of six CDRs (vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2 and vlCDR3) fromCHA.9.547.13.H4(S241P) as depicted in FIG. 3 . In one embodiment, theanti-TIGIT antibody is an antibody comprising the variable heavy (vh)and variable light (vl) domains from CHA.9.547.13.H4(S241P) as depictedin FIG. 3 , linked to a human IgG constant domain of IgG1, IgG2, IgG3,IgG4 and IgG4(S241P). In one embodiment, the anti-TIGIT antibody isCHA.9.547.13.H4(S241P).

Further anti-TIGIT antibodies that find use in combinations withanti-PVRIG antibodies as outlined herein are those in FIG. 4 of U.S.Ser. No. 62/513,916, entitled “Anti-TIGIT Antibodies and Methods ofUse”, filed on Jun. 1, 2017, by assignee Compugen, as well as thoseincluded in FIG. 3 .

C. Additional Anti-TIGIT Antibodies for Use in Combination Therapy

Additional anti-TIGIT antibodies that can be used in combination withanti-PVRIG antibodies and optionally anti-PD-1 antibodies as outlinedherein are also included. As discussed more fully below, anti-TIGITantibodies show particular efficacy in combination with anti-PVRIGantibodies. Thus, in some embodiments, alternative anti-TIGIT antibodiesare used in combination with anti-PVRIG antibodies outlined herein, andin particular either of CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P).

Accordingly, in one embodiment, anti-TIGIT antibodies as outlined inU.S. Pat. No. 9,499,596, (hereby incorporated by reference in itsentirety and specifically for the SEQ ID NO:s listed below) can becombined with CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P).Specifically, an anti-TIGIT antibody having a light chain sequence ofSEQ ID NO:21 and a heavy chain sequence of SEQ ID NO:22 (from U.S. Pat.No. 9,499,596) can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Additionally, an anti-TIGIT antibody having alight chain sequence of SEQ ID NO:29 and a heavy chain sequence of SEQID NO:30 (from U.S. Pat. No. 9,499,596) can be combined withCHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P).

Similarly, in one embodiment, anti-TIGIT antibodies as outlined in WO2016/191643 (hereby incorporated by reference in its entirety andspecifically for the SEQ ID NO:s listed below, and in particular for thesequences of the OMP-313M32 antibody) can be combined withCHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Specifically, ananti-TIGIT antibody having a light chain sequence of SEQ ID NO:72 and aheavy chain sequence of SEQ ID NO:70 (from WO 2016/191643) can becombined with CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P).

Accordingly, in one embodiment, anti-TIGIT antibodies as outlined in WO2017/053748 (hereby incorporated by reference in its entirety andspecifically for the SEQ ID NO:s listed below) can be combined withCHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Specifically, ananti-TIGIT antibody having a variable light chain sequence of SEQ IDNO:36 and a variable heavy chain sequence of SEQ ID NO:34 (from WO2017/053748) can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Additionally, an anti-TIGIT antibody having avariable light chain sequence of SEQ ID NO:36 and a variable heavy chainsequence of SEQ ID NO:35 (from WO 2017/053748) can be combined withCHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Additionally, ananti-TIGIT antibody having a variable light chain sequence of SEQ IDNO:38 and a variable heavy chain sequence of SEQ ID NO:37 (from WO2017/053748) can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Additionally, an anti-TIGIT antibody having avariable light chain sequence of SEQ ID NO:40 and a variable heavy chainsequence of SEQ ID NO:39 (from WO 2017/053748) can be combined withCHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). In one embodiment,anti-TIGIT antibodies include the Genentech antibody, MTIG7192A,currently in clinical trials (see, the World Wide Web atclinicaltrials.gov/ct2/show/NCT02794571?term=MTIG7192M&rank=1). In oneembodiment, an MTIG7192A anti-TIGIT antibody can be combined withCHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P).

Similarly, in one embodiment, anti-TIGIT antibodies as outlined inWO2016/191643 (hereby incorporated by reference in its entirety andspecifically for the SEQ ID NO:s listed below, and in particular for thesequences of the OMP-313M32 antibody can be combined withCHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Specifically, ananti-TIGIT antibody having a light chain sequence of SEQ ID NO:72 and aheavy chain sequence of SEQ ID NO:70 (from WO2016/191643) can becombined with CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). In oneembodiment, anti-TIGIT antibodies include the Oncomed antibody,OMP-313M32, currently in clinical trials (see, the World Wide Web atclinicaltrials.gov/ct2/show/NCT03119428?term=OMP-313M32&rank=1). In oneembodiment, an OMP-313M32 anti-TIGIT antibody can be combined withCHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P).

Additionally, in one embodiment, anti-TIGIT antibodies as outlined in WO2016/028656 (hereby incorporated by reference in its entirety andspecifically for the SEQ ID NO:s listed below, and in particular for thesequences of the MEB125.31C6.A1.205 VH4/VL1 (VH of SEQ ID NO:127, VL ofSEQ ID NO:130 with a human IgG1 constant domain), MEB 125.3106.A1.205VH5/VL4 (VH of SEQ ID NO:128, VL of SEQ ID NO:133 and a human IgG1constant region) and MEB125.31.C6,A1.205 VH5/VL3 (VH of SEQ ID NO:128,VL of SEQ ID NO:132 and a human IgG1 constant region) antibodies) can becombined with CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P).Specifically, an anti-TIGIT antibody MEB125.31C6.A1.205 VH4/VL1 (VH ofSEQ ID NO:127, VL of SEQ ID NO:130 with a human IgG1 constant domain)(from WO 2016/028656) can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Specifically, an anti-TIGIT antibody MEB125.3106.A1.205 VH5/VL4 (VH of SEQ ID NO:128, VL of SEQ ID NO:133 and ahuman IgG1 constant region (from WO 2016/028656) can be combined withCHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Specifically, ananti-TIGIT antibody MEB125.31.C6.A1.205 VH5NL3 (VH of SEQ ID NO:128, VLof SEQ ID NO:132 and a human IgG1 constant region) (from WO2016/028656)can be combined with CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P).Specifically, an anti-TIGIT antibody comprising the VH of SEQ ID NO:7,VL of SEQ ID NO:8 and a human IgG1 constant region (from WO 2016/028656)can be combined with CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P).Specifically, an anti-TIGIT antibody comprising the VH of SEQ ID NO:63,VL of SEQ ID NO:64 and a human IgG1 constant region (from WO2016/028656) can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Specifically, an anti-TIGIT antibody comprisingthe VH of SEQ ID NO:94, VL of SEQ ID NO:95 and a human IgG1 constantregion (from WO 2016/028656) can be combined withCHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Specifically, ananti-TIGIT antibody comprising the VH of SEQ ID NO:126, VL of SEQ IDNO:131 and a human IgG1 constant region (from WO 2016/028656) can becombined with CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P).Specifically, an anti-TIGIT antibody comprising the VH of SEQ ID NO:128,VL of SEQ ID NO:131 and a human IgG1 constant region (from WO2016/028656) can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Specifically, an anti-TIGIT antibody comprisingthe VH of SEQ ID NO:125, VL of SEQ ID NO:133 and a human IgG1 constantregion (from WO 2016/028656) can be combined withCHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Specifically, ananti-TIGIT antibody comprising the VH of SEQ ID NO:126, VL of SEQ IDNO:130 and a human IgG1 constant region (from WO 2016/028656) can becombined with CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P).Specifically, an anti-TIGIT antibody comprising the VH of SEQ ID NO:125,VL of SEQ ID NO:132 and a human IgG1 constant region (from WO2016/028656) can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Specifically, an anti-TIGIT antibody comprisingthe VH of SEQ ID NO:143, VL of SEQ ID NO:145 and a human IgG1 constantregion (from WO 2016/028656) can be combined withCHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Specifically, ananti-TIGIT antibody comprising the VH of SEQ ID NO:144, VL of SEQ IDNO:146 and a human IgG1 constant region (from WO 2016/028656) can becombined with CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P).Specifically, an anti-TIGIT antibody comprising the VH of SEQ ID NO:149,VL of SEQ ID NO:151 and a human IgG1 constant region (from WO2016/028656) can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Specifically, an anti-TIGIT antibody comprisingthe VH of SEQ ID NO:150, VL of SEQ ID NO:152 and a human IgG1 constantregion (from WO 2016/028656) can be combined withCHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P).

Additionally, in one embodiment, anti-TIGIT antibodies as outlined in WO2017/030823 hereby incorporated by reference in its entirety andspecifically for the SEQ ID NO:s listed below) can be combined withCHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Specifically, ananti-TIGIT antibody having a variable light chain sequence of SEQ IDNO:8 and a variable heavy chain sequence of SEQ ID NO:7 (from WO2017/030823) can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Specifically, an anti-TIGIT antibody having avariable light chain sequence of SEQ ID NO:13 and a variable heavy chainsequence of SEQ ID NO:9 (from WO 2017/030823) can be combined withCHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Specifically, ananti-TIGIT antibody having a variable light chain sequence of SEQ IDNO:24 and a variable heavy chain sequence of SEQ ID NO:23 (from WO2017/030823) can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Specifically, an anti-TIGIT antibody having avariable light chain sequence of SEQ ID NO:29 and a variable heavy chainsequence of SEQ ID NO:25 (from WO 2017/030823) can be combined withCHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). In some embodiments,an anti-TIGIT antibody having a variable light chain selected fromsequences of SEQ ID NO:s 14, 15, 16, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74 ,75, 76, 77, 78, 79, or 80 and a variable heavy chainsequence of SEQ ID NO:s 10, 11 ,12, 48, 49, 50, 51, 52, 53, 54, 55, or56 (from WO 2017/030823) can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). In some embodiments, an anti-TIGIT antibodyhaving a variable light chain selected from sequences of SEQ ID NO:s 14,15, 16, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 ,75, 76, 77, 78,79, or 80 and a variable heavy chain sequence of SEQ ID NO:s 10, 11 ,12,48, 49, 50, 51, 52, 53, 54, 55, or 56 (from WO2017/030823) can becombined with CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). In someembodiments, an anti-TIGIT antibody having a variable light chainselected from sequences of SEQ ID NO:s 30, 31, or 32 and a variableheavy chain sequence of SEQ ID NO: 26, 27, 28, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, or 112 (from WO2017/030823) can be combined withCHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). In one embodiment,anti-TIGIT antibodies include the Merck antibody, MK-7684, currently inclinical trials (see, the World Wide Web atclinicaltrials.gov/ct2/show/NCT02964013?term=MK-7684&rank=1). In oneembodiment, an MK-7684 anti-TIGIT antibody can be combined withCHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P).

Accordingly, in one embodiment, anti-TIGIT antibodies as outlined in USPatent Appl. No. 2016/0176963 (hereby incorporated by reference in itsentirety and specifically for the SEQ ID NO:s listed below) can becombined with CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P).Specifically, an anti-TIGIT antibody having a variable light chainsequence of SEQ ID NO:s 6, 9, 11, or 13 and a variable heavy chainsequence of SEQ ID NO:s 2, 3, 4, 5, 7, 8, 10, or 12 (fromUS2016/0176963) can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Specifically, an anti-TIGIT antibody having avariable light chain sequence of SEQ ID NO:s 6 and a variable heavychain sequence of SEQ ID NO:s 2, 3, 4, or 5 (from US2016/0176963) can becombined with CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P).Specifically, an anti-TIGIT antibody having a variable light chainsequence of SEQ ID NO:s 9 and a variable heavy chain sequence of SEQ IDNO:s 7 or 8 (from US2016/0176963) can be combined withCHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Specifically, ananti-TIGIT antibody having a variable light chain sequence of SEQ ID NO:11 and a variable heavy chain sequence of SEQ ID NO: 10 (fromUS2016/0176963) can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Specifically, an anti-TIGIT antibody having avariable light chain sequence of SEQ ID NO: 13 and a variable heavychain sequence of SEQ ID NO: 12 (from US2016/0176963) can be combinedwith CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). In oneembodiment, anti-TIGIT antibodies include the BMS antibody, BMS-98620,currently in clinical trials (see, the World Wide Web atclinicaltrials.gov/ct2/show/NCT02913313?term=BMS-986207&rank=1). In oneembodiment, an BMS-98620 anti-TIGIT antibody can be combined withCHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P).

In one embodiment, anti-TIGIT antibodies include the Arcus Bio antibody,AB154. In one embodiment, an AB154 anti-TIGIT antibody can be combinedwith CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P).

Additionally, in one embodiment, anti-TIGIT antibodies as outlined in WO2017/037707 (hereby incorporated by reference in its entirety andspecifically for the SEQ ID NO:s listed below, and in particular for thesequences of the SEQ ID NO:s listed below, and in particular for theVSIG9#1 antibody (SEQ ID NO:7 VH and SEQ ID NO: 8 VL) and the 258-csl#4antibody (SEQ ID NO:18 VH and SEQ ID NO: 19 VL). Specifically, theanti-TIGIT antibody VSIG9#1 antibody (from WO2017/037707; SEQ ID NO:7 VHand SEQ ID NO: 8 VL) can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Specifically, the anti-TIGIT antibody 258-csl#4(from WO2017/037707; SEQ ID NO:18 VH and SEQ ID NO: 19 VL) can becombined with CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P).

Additionally, in one embodiment, anti-TIGIT antibodies as outlined in WO2017/059095 (hereby incorporated by reference in its entirety andspecifically for the SEQ ID NO:s listed below, and in particular for thesequences disclosed therein. Specifically, the anti-TIGIT antibodycomprising a VH sequence of SEQ ID NO: 13 and a V_(L) sequence of SEQ IDNO: 26 (from WO 2017/059095) can be combined withCHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Specifically, theanti-TIGIT antibody comprising a VH sequence of SEQ ID NO: 12 and aV_(L) sequence of SEQ ID NO: 26 (from WO 2017/059095) can be combinedwith CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Specifically, theanti-TIGIT antibody comprising a VH sequence of SEQ ID NO: 14 and aV_(L) sequence of SEQ ID NO: 26 (from WO 2017/059095) can be combinedwith CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Specifically, theanti-TIGIT antibody comprising a VH sequence of SEQ ID NO: 15 and aV_(L) sequence of SEQ ID NO: 26 (from WO 2017/059095) can be combinedwith CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Specifically, theanti-TIGIT antibody comprising a VH sequence of SEQ ID NO: 9 and a VLsequence of SEQ ID NO: 26 (from WO 2017/059095) can be combined withCHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Specifically, theanti-TIGIT antibody comprising a VH sequence of SEQ ID NO: 10 and a VLsequence of SEQ ID NO: 26 (from WO 2017/059095) can be combined withCHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Specifically, theanti-TIGIT antibody comprising a VH sequence of SEQ ID NO: 1 1 and a VLsequence of SEQ ID NO: 26 (from WO 2017/059095) can be combined withCHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Specifically, theanti-TIGIT antibody comprising a heavy chain of SEQ ID NO: 99 and alight chain of SEQ ID NO: 92; or (it) a heavy chain of SEQ ID NO: 100and a light chain of SEQ ID NO: 92 (from WO 2017/059095) can be combinedwith CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Specifically, theanti-TIGIT antibody comprising a heavy chain of SEQ ID NO: 97 and alight chain of SEQ ID NO: 92; or (ii) a heavy chain of SEQ ID NO: 98 anda light chain of SEQ ID NO: 92 (from WO 2017/059095) can be combinedwith CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Specifically, theanti-TIGIT antibody comprising a heavy chain of SEQ ID NO: 101 and alight chain of SEQ ID NO: 92; or (ii) a heavy chain of SEQ ID NO: 102and a light chain of SEQ ID NO: 92 (from WO 2017/059095) can be combinedwith CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Specifically, theanti-TIGIT antibody comprising a heavy chain of SEQ ID NO: 103 and alight chain of SEQ ID NO: 92; or (ii) a heavy chain of SEQ ID NO: 104and a light chain of SEQ ID NO: 92 (from WO 2017/059095) can be combinedwith CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Specifically, theanti-TIGIT antibody comprising a heavy chain of SEQ ID NO: 90 and alight chain of SEQ ID NO: 92; or (ii) a heavy chain of SEQ ID NO: 91 anda light chain of SEQ ID NO: 92 (from WO 2017/059095) can be combinedwith CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Specifically, theanti-TIGIT antibody comprising a heavy chain of SEQ ID NO: 93 and alight chain of SEQ ID NO: 92; or (ii) a heavy chain of SEQ ID NO: 94 anda light chain of SEQ ID NO: 92 (from WO 2017/059095) can be combinedwith CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Specifically, theanti-TIGIT antibody comprising a heavy chain of SEQ ID NO: 95 and alight chain of SEQ ID NO: 92; or (ii) a heavy chain of SEQ ID NO: 96 anda light chain of SEQ ID NO: 92 (from WO 2017/059095) can be combinedwith CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P).Additionally, inone embodiment, anti-TIGIT antibodies as outlined in WO 2016/106302(hereby incorporated by reference in its entirety and specifically forthe SEQ ID NO:s listed below, and in particular for the sequences of theparticular sequences disclosed therein. Specifically, the anti-TIGITantibody comprising a heavy chain sequence selected from SEQ ID NOs: 2,3, 4, 5, 7, 8, 10 or 12 (from WO 2016/106302) and a light chain sequencefrom SEQ ID NOs: 6, 9, 11, or 13 (from WO 2016/106302) can be combinedwith CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Specifically, theanti-TIGIT antibody 22G2 (from WO 2016/106302) can be combined withCHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Specifically, theanti-TIGIT antibody 11G11 (from WO 2016/106302) can be combined withCHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Specifically, theanti-TIGIT antibody 15A6 (from WO 2016/106302) can be combined withCHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P).

Additionally, in one embodiment, anti-TIGIT antibodies as outlined inU.S. Patent Publication No. 2017281764 (hereby incorporated by referencein its entirety and specifically for the SEQ ID NO:s listed below, andin particular for the sequences disclosed therein. Specifically, theanti-TIGIT antibody comprising a V_(H) sequence of SEQ ID NO: 10 and aV_(L) sequence of SEQ ID NO: 14 (from US 2017281764) can be combinedwith CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Specifically, theanti-TIGIT antibody comprising a V_(H) sequence of SEQ ID NO: 18 and aV_(L) sequence of SEQ ID NO: 22 (from US 2017281764) can be combinedwith CHA.7.538.1.2.H4(S241P) or CHA. 7.518.1.H4(S241P). Specifically,the anti-TIGIT antibody comprising a V_(H) sequence of SEQ ID NO: 26 anda V_(L) sequence of SEQ ID NO: 30 (from US 2017281764) can be combinedwith CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Specifically, theanti-TIGIT antibody comprising a V_(H) sequence of SEQ ID NO: 35 and aV_(L) sequence of SEQ ID NO: 37 (from US 2017281764) can be combinedwith CHA.7.538.1.2.H4(S241P) or CHA. 7.518.1.H4(S241P). Specifically,the anti-TIGIT antibody comprising a V_(H) sequence of SEQ ID NO: 34 anda V_(L) sequence of SEQ ID NO: 36 (from US 2017281764) can be combinedwith CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P).

In another embodiment, anti-TIGIT antibodies as outlined inInternational Patent Publication No. WO 2015/009856 (hereby incorporatedby reference in its entirety and specifically for the SEQ ID NO:s listedbelow, and in particular for the sequences disclosed therein (see, alsoInternational Patent Publication No. WO 2016/011264). Specifically, theanti-TIGIT antibody comprising a VH sequence of SEQ ID NO: 15 and aV_(L) sequence of SEQ ID NO: 13 (from WO 2015/009856) can be combinedwith CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Specifically, theanti-TIGIT antibody comprising a VH sequence of SEQ ID NO: 16 and aV_(L) sequence of SEQ ID NO: 14 (from WO 2015/009856) can be combinedwith CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P).

In some embodiments, the anti-TIGIT antibody is an antibody described inany of U.S. Patent Application No. 20170037133, International PatentPublication No. WO 2017/048824, a MBSA43 (commercially available fromeBioscience), is anti-TIGIT antibody pab2197 or pab2196 (U.S. PatentApplication No. 2017/0081409), E05084448, CASC-674 (available fromAdimab LLC). Specifically, an anti-TIGIT antibody as described in U.S.Patent Application No. 2017/0037133 can be combined withCHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Specifically, ananti-TIGIT antibody as described in International Patent Publication No.WO 2017/048824 can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Specifically, the anti-TIGIT antibody MBSA43 canbe combined with CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P).Specifically, the anti-TIGIT antibody pab2197 can be combined withCHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Specifically, theanti-TIGIT antibody pab2196 can be combined with CHA.7.538.1.2.H4(S241P)or CHA.7.518.1.H4(S241P). Specifically, the anti-TIGIT antibodyE05084448 can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Specifically, the anti-TIGIT antibody CASC-674can be combined with CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P).

In some embodiments, the anti-TIGIT antibody is an antibody described inU.S. Pat. No. 9,713,364 (incorporated herein by reference in itsentirety). In some embodiments, the anti-TIGIT antibody is PTZ-201(ASP8374). Specifically, the anti-TIGIT antibody PTZ-201 (ASP8374) canbe combined with CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Insome embodiments, the anti-TIGIT antibody is an antibody selected fromthe group consisting of MAB1, MAB2, MAB3, MAB4, MAB5, MAB6, MAB7, MAB8,MABg, MAB10, MAB11, AB12, MAB13, MAB14, MAB15, AB16, MAB17, MAB18, 40MAB19, MAB20, or MAB21, as described in U.S. Pat. No. 9,713,364.Specifically, the anti-TIGIT antibody MAB1 from U.S. Pat. No. 9,713,364can be combined with CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P).Specifically, the anti-TIGIT antibody MAB2 from U.S. Pat. No. 9,713,364can be combined with CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P).Specifically, the anti-TIGIT antibody MAB3 from U.S. Pat. No. 9,713,364can be combined with CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P).Specifically, the anti-TIGIT antibody MAB4 from U.S, Patent No,9,713,364 can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Specifically, the anti-TIGIT antibody MAB3 fromU.S. Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Specifically, the anti-TIGIT antibody MAB6 fromU.S. Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Specifically, the anti-TIGIT antibody MAB7 fromU.S. Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Specifically, the anti-TIGIT antibody MAB8 fromU.S. Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Specifically, the anti-TIGIT antibody MAB fromU.S. Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Specifically, the anti-TIGIT antibody MAB10 fromU.S. Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Specifically, the anti-TIGIT antibody MAB11 fromU.S. Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Specifically, the anti-TIGIT antibody MAB12 fromU.S. Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Specifically, the anti-TIGIT antibody MAB13 fromU.S. Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Specifically, the anti-TIGIT antibody MAB14 fromU.S. Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Specifically, the anti-TIGIT antibody MAB15 fromU.S. Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Specifically, the anti-TIGIT antibody MAB1 6 fromU.S. Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Specifically, the anti-TIGIT antibody MAB17 fromU.S. Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Specifically, the anti-TIGIT antibody MAB18 fromU.S. Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Specifically, the anti-TIGIT antibody MAB19 fromU.S. Pat. No, 9,713,364 can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Specifically, the anti-TIGIT antibody MAB20 fromU.S. Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Specifically, the anti-TIGIT antibody MAB21 fromU.S. Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P).

In some embodiments, the anti-TIGIT antibody is 10A7, 1F4, 14A6, 28H5,3106, 15A6, 22G2, 11G11, and/or 10D7, the contents of each of which areincorporated herein by reference in their entirety. Specifically, theanti-TIGIT antibody 10A7 can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Specifically, the anti-TIGIT antibody 1F4 can becombined with CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P).Specifically, the anti-TIGIT antibody 14A6 can be combined withCHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Specifically, theanti-TIGIT antibody 28H5 can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Specifically, the anti-TIGIT antibody 3106 can becombined with CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P).Specifically, the anti-TIGIT antibody 15A6 can be combined withCHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P). Specifically, theanti-TIGIT antibody 22G2 can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P). Specifically, the anti-TIGIT antibody 11G11 canbe combined with CHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P).Specifically, the anti-TIGIT antibody 10D7 can be combined withCHA.7.538.1.2.H4(S241P) or CHA.7.518.1.H4(S241P).

In some embodiments, the anti-TIGIT antibody is one of those describedin International Patent Publication WO 2016/028656, incorporated hereinin its entirety. Specifically, the anti-TIGIT antibodies are provided inInternational Patent Publication No. WO 2016/028656 (and reproducedherein in FIG. 3 ) can be combined with CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P).

The anti-TIGIT antibodies described herein can find use according to thetriple combination therapy methods of the invention are labeled asfollows. Such TIGIT antibodies have reference numbers, for example“CPA.9.086”. This represents the combination of the variable heavy andvariable light chains, as depicted in FIG. 3 , for example, with theunderstanding that these antibodies include two heavy chains and twolight chains. “CPA.9.086.VH” refers to the variable heavy portion ofCPA.9. 086, while “CPA.9. 086.VL” is the variable light chain. “CPA.9.086.vhCDR1”, “CPA.9. 086.vhCDR2”, “CPA. 9. 086.vhCDR3”, “CPA.9.086.vlCDR1”, “CPA.9. 086.vlCDR2”, and “CPA.9. 086.vlCDR3”, refers to theCDRs are indicated. “CPA.9. 086.HC” refers to the entire heavy chain(e.g. variable and constant domain) of this molecule, and “CPA.9.086.LC” refers to the entire light chain (e.g. variable and constantdomain) of the same molecule. In general, the human kappa light chain isused for the constant domain of each phage (or humanized hybridoma)antibody herein, although in some embodiments the lambda light constantdomain is used. “CPA.9. 086.H1” refers to a full length antibodycomprising the variable heavy and light domains, including the constantdomain of Human IgG1 (hence, the H1; IgG1, IgG2, IgG3 and IgG4 sequencesare shown in FIGS. 1 , for example). Accordingly, “CPA.9. 086.H2” wouldbe the CPA.9. 086 variable domains linked to a Human IgG2. “CPA.9.086.H3” would be the CPA.9. 086 variable domains linked to a Human IgG3,and “CPA.9. 086.H4” would be the CPA.9. 086 variable domains linked to aHuman IgG4. Note that in some cases, the human IgGs may have additionalmutations, such are described below, and this can be annotated. Forexample, in many embodiments, there may be a S241P mutation in the humanIgG4, and this can be annotated as “CPA.9.086.H4(S241P)” for example.The human IgG4 sequence with this S241P hinge variant is shown in FIG. 1. Other potential variants are IgG1(N297A), (or other variants thatablate glycosylation at this site and thus many of the effectorfunctions associated with FcγRIIIa binding), and IgG1(D265A), whichreduces binding to FcγR receptors. The anti-TIGIT antibodies for use inthe present invention can comprise any of the TIGIT antibody domainsequences. The anti-TIGIT antibodies for use in the present inventioncan comprise any of the TIGIT antigen binding domains.

The invention further provides variable heavy and light domains as wellas full length heavy and light chains.

In some embodiments, the invention provides scFvs that bind to TIGITcomprising a variable heavy domain and a variable light domain linked byan scFv linker as outlined above. The VL and VH domains can be in eitherorientation, e.g. from N- to C-terminus “VH-linker-VL” or “VL-linker”VH”. These are named by their component parts; for example,“scFv-CPA.9.086.VH-linker-VL” or “scFv-CPA.9.086.VL-linker-VH.” Thus,“scFv-CPA.9.086” can be in either orientation. The anti-TIGIT antibodiesfor use in the present invention can comprise any scFvs that bind toTIGIT. The anti-TIGIT antibodies for use in the present invention cancomprise any scFvs that bind to TIGIT. The anti-TIGIT antibodies for usein the present invention can include any of the following:

CPA.9.018, CPA.9.018.VH, CPA.9.018.VL, CPA.9.018.HC, CPA.9.018.LC,CPA.9.018.H1, CPA.9.018.H2, CPA.9.018.H3, CPA.9.018.H4;CPA.9.018.H4(S241P); CPA.9.018.vhCDR1, CPA.9.018.vhCDR2,CPA.9.018.vhCDR3, CPA.9.018.vlCDR1, CPA.9.018.vlCDR2, CPA.9.018.vlCDR3and scFv-CPA.9.018;

CPA.9.027, CPA.9.027.VH, CPA.9.027.VL, CPA.9.027.HC, CPA.9.027.LC,CPA.9.027.H1, CPA.9.027.H2, CPA.9.027.H3, CPA.9.027.H4;CPA.9.018.H4(S241P); CPA.9.027.vhCDR1, CPA.9.027.vhCDR2,CPA.9.027.vhCDR3, CPA.9.027.vlCDR1, CPA.9.027.vlCDR2, CPA.9.027.vlCDR3and scFv-CPA.9.027;

CPA.9.049, CPA.9.049.VH, CPA.9.049.VL, CPA.9.049.HC, CPA.9.049.LC,CPA.9.049.H1, CPA.9.049.H2, CPA.9.049.H3; CPA.9.049.H4;CPA.9.049.H4(S241P); CPA.9.049.vhCDR1, CPA.9.049.vhCDR2,CPA.9.049.vhCDR3, CPA.9.049.vlCDR1, CPA.9.049.vlCDR2, CPA.9.049.vlCDR3and scFv-CPA.9.049;

CPA.9.057, CPA.9.057.VH, CPA.9.057.VL, CPA.9.057.HC, CPA.9.057.LC,CPA.9.057.H1, CPA.9.057.H2, CPA.9.057.H3; CPA.9.057.H4;CPA.9.057.H4(S241P); CPA.9.057.vhCDR1, CPA.9.057.vhCDR2,CPA.9.057.vhCDR3, CPA.9.057.vlCDR1, CPA.9.057.vlCDR2, CPA.9.057.vlCDR3and scFv-CPA.9.057;

CPA.9.059, CPA.9.059.VH, CPA.9.059.VL, CPA.9.059.HC, CPA.9.059.LC,CPA.9.059.H1, CPA.9.059.H2, CPA.9.059.H3; CPA.9.059.H4;CPA.9.059.H4(S241P); CPA.9.059.vhCDR1, CPA.9.059.vhCDR2,CPA.9.059.vhCDR3, CPA.9.059.vlCDR1, CPA.9.059.vlCDR2, CPA.9.059.vlCDR3and scFv-CPA.9.059;

CPA.9.083, CPA.9.083.VH, CPA.9.083.VL, CPA.9.083.HC, CPA.9.083.LC,CPA.9.083.H1, CPA.9.083.H2, CPA.9.083.H3; CPA.9.083.H4;CPA.9.083.H4(S241P); CPA.9.083.vhCDR1, CPA.9.083.vhCDR2,CPA.9.083.vhCDR3, CPA.9.083.vlCDR1, CPA.9.083.vlCDR2, CPA.9.083.vlCDR3and scFv-CPA.9.083;

CPA.9.086, CPA.9.086.VH, CPA.9.086.VL, CPA.9.086.HC, CPA.9.086.LC,CPA.9.086.H1, CPA.9.086.H2, CPA.9.086.H3; CPA.9.086.H4;CPA.9.086.H4(S241P); CPA.9.086.vhCDR1, CPA.9.086.vhCDR2,CPA.9.086.vhCDR3, CPA.9.086.vlCDR1, CPA.9.086.vlCDR2, CPA.9.086.vlCDR3and scFv-CPA.9.086;

CPA.9.089, CPA.9.089.VH, CPA.9.089.VL, CPA.9.089.HC, CPA.9.089.LC,CPA.9.089.H1, CPA.9.089.H2, CPA.9.089.H3; CPA.9.089.H4;CPA.9.089.H4(S241P); CPA.9.089.vhCDR1, CPA.9.089.vhCDR2,CPA.9.089.vhCDR3, CPA.9.089.vlCDR1, CPA.9.089.vlCDR2, CPA.9.089.vlCDR3and scFv-CPA.9.089;

CPA.9.093, CPA.9.093.VH, CPA.9.093.VL, CPA.9.093 .HC, CPA.9.093 .LC,CPA.9.093.H1, CPA.9.093.H2, CPA.9.093.H3; CPA.9.093.H4;CPA.9.093.H4(S241P); CPA.9.093.vhCDR1, CPA.9.093.vhCDR2,CPA.9.093.vhCDR3, CPA.9.093.vlCDR1, CPA.9.093.vlCDR2, CPA.9.093.vlCDR3and scFv-CPA.9.093;

CPA.9.101, CPA.9.101.VH, CPA.9.101.VL, CPA.9.101.HC, CPA.9.101.LC,CPA.9.101.H1, CPA.9.101.H2, CPA.9.101.H3; CPA.9.101.H4;CPA.9.101.H4(S241P); CPA.9.101.vhCDR1, CPA.9.101.vhCDR2,CPA.9.101.vhCDR3, CPA.9.101.vlCDR1, CPA.9.101.vlCDR2, CPA.9.101.vlCDR3and scFv-CPA.9.101; and

CPA.9.103, CPA.9.103.VH, CPA.9.103.VL, CPA.9.103.HC, CPA.9.103.LC,CPA.9.103.H1, CPA.9.103.H2, CPA.9.103.H3; CPA.9.103.H4;CPA.9.103.H4(S241P); CPA.9.103.vhCDR1, CPA.9.103.vhCDR2,CPA.9.103.vhCDR3, CPA.9.103.vlCDR1, CPA.9.103.vlCDR2, CPA.9.103.vlCDR3and scFv-CPA.9.103.

Furthermore, the present invention provides a number of CHA antibodies,which are murine antibodies generated from hybridomas. As is well knownthe art, the six CDRs are useful when put into either human frameworkvariable heavy and variable light regions or when the variable heavy andlight domains are humanized. Accordingly, the present invention providesantibodies, usually full length or scFv domains, that comprise thefollowing sets of CDRs, the sequences of which are shown in FIG. 3and/or the sequence listing:

CHA.9.536.1, CHA.9.536.1.VH, CHA.9.536.1.VL, CHA.9.536.1.HC,CHA.9.536.1.LC, CHA.9.536.1.H1, CHA.9.536.1.H2, CHA.9.536.1.H3;CHA.9.536.1.H4, CHA.9.536.1.H4(S241P), CHA.9.536.1.vhCDR1,CHA.9.536.1.vhCDR2, CHA.9.536.1.vhCDR3, CHA.9.536.1.vlCDR1,CHA.9.536.1.vlCDR2 and CHA.9.536.1.vhCDR3;

CHA.9.536.3, CHA.9.536.3.VH, CHA.9.536.3.VL, CHA.9.536.3.HC,CHA.9.536.3.LC, CHA.9.536.3.H1, CHA.9.536.3.H2, CHA.9.536.3.H3;CHA.9.536.3.H4, CHA.9.536.3.H4(S241P); CHA.9.536.3.vhCDR1,CHA.9.536.3.vhCDR2, CHA.9.536.3.vhCDR3, CHA.9.536.3.vlCDR1,CHA.9.536.3.vlCDR2 and CHA.9.536.3.vhCDR3;

CHA.9.536.4, CHA.9.536.4.VH, CHA.9.536.4.VL, CHA.9.536.4.HC,CHA.9.536.4.LC, CHA.9.536.4.H1, CHA.9.536.4.H2, CHA.9.536.4.H3;CHA.9.536.4.H4, CHA.9.536.4.H4(S241P), CHA.9.536.4.vhCDR1,CHA.9.536.4.vhCDR2, CHA.9.536.4.vhCDR3, CHA.9.536.4.vlCDR1,CHA.9.536.4.vlCDR2 and CHA.9.536.4.vhCDR3;

CHA.9.536.5, CHA.9.536.5.VH, CHA.9.536.5.VL, CHA.9.536.5.HC,CHA.9.536.5.LC, CHA.9.536.5.H1, CHA.9.536.5.H2, CHA.9.536.5.H3;CHA.9.536.5.H4, CHA.9.536.5.H4(S241P), CHA.9.536.5.vhCDR1,CHA.9.536.5.vhCDR2, CHA.9.536.5.vhCDR3, CHA.9.536.5.vlCDR1,CHA.9.536.5.vlCDR2 and CHA.9.536.5.vhCDR3;

CHA.9.536.6, CHA.9.536.6.VH, CHA.9.536.6.VL, CHA.9.536.6.HC,CHA.9.536.6.LC, CHA.9.536.6.H1, CHA.9.536.6.H2, CHA.9.536.6.H3;CHA.9.536.6.H4, CHA.9.536.6.vhCDR1, CHA.9.536.6.vhCDR2,CHA.9.536.6.vhCDR3, CHA.9.536.6.vlCDR1, CHA.9.536.6.vlCDR2 andCHA.9.536.6.vhCDR3;

CHA.9.536.7, CHA.9.536.7.VH, CHA.9.536.7.VL, CHA.9.536.7.HC,CHA.9.536.7.LC, CHA.9.536.7.H1, CHA.9.536.7.H2, CHA.9.536.7.H3;CHA.9.536.7.H4, CHA.9.536.5.H4(S241P); CHA.9.536.7.vhCDR1,CHA.9.536.7.vhCDR2, CHA.9.536.7.vhCDR3, CHA.9.536.7.vlCDR1,CHA.9.536.7.vlCDR2 and CHA.9.536.7.vhCDR3;

CHA.9.536.8, CHA.9.536.8.VH, CHA.9.536.8.VL, CHA.9.536.8.HC,CHA.9.536.8.LC, CHA.9.536.8.H1, CHA.9.536.8.H2, CHA.9.536.8.H3;CHA.9.536.8.H4, CHA.9.536.8.H4(S241P), CHA.9.536.8.vhCDR1,CHA.9.536.8.vhCDR2, CHA.9.536.8.vhCDR3, CHA.9.536.8.vlCDR1,CHA.9.536.8.vlCDR2 and CHA.9.536.8.vhCDR3;

CHA.9.560.1, CHA. 9.560.1VH, CHA. 9.560.1.VL, CHA. 9.560.1.HC, CHA.9.560.1.LC, CHA. 9.560.1.H1, CHA. 9.560.1.H2, CHA. 9.560.1.H3; CHA.9.560.1.H4, CHA. 9.560.1.H4(S241P), CHA. 9.560.1.vhCDR1, CHA.9.560.1.vhCDR2, CHA. 9.560.1.vhCDR3, CHA. 9.560.1.vlCDR1, CHA.9.560.1.vlCDR2 and CHA. 9.560.1.vhCDR3;

CHA.9.560.3, CHA. 9.560. 3VH, CHA. 9.560. 3.VL, CHA. 9.560. 3.HC, CHA.9.560. 3.LC, CHA. 9.560. 3.H1, CHA. 9.560. 3.H2, CHA. 9.560. 3.H3;CHA.9.560.3.H4, CHA.9.560.3.H4(S241P); CHA. 9.560. 3.vhCDR1, CHA. 9.560.3.vhCDR2, CHA. 9.560. 3.vhCDR3, CHA. 9.560. 3.vlCDR1, CHA. 9.560.3.vlCDR2 and CHA. 9.560. 3.vhCDR3;

CHA.9.560.4, CHA. 9.560. 4VH, CHA. 9.560. 4.VL, CHA. 9.560. 4.HC, CHA.9.560. 4.LC, CHA. 9.560. 4.H1, CHA. 9.560. 4.H2, CHA. 9.560. 4.H3;CHA.9.560.4.H4, CHA.9.560.4.H4(S241P), CHA. 9.560. 4.vhCDR1, CHA. 9.560.4.vhCDR2, CHA. 9.560. 4.vhCDR3, CHA. 9.560. 4.vlCDR1, CHA. 9.560.4.vlCDR2 and CHA. 9.560. 4.vhCDR3;

CHA.9.560.5, CHA. 9.560. SVH, CHA. 9.560. 5.VL, CHA. 9.560. 5.HC, CHA.9.560. 5.LC, CHA. 9.560. 5.H1, CHA. 9.560. 5.H2, CHA. 9.560. 5.H3; CHA.9.560. 5.H4, CHA. 9.560. 5.vhCDR1, CHA. 9.560. 5.vhCDR2, CHA. 9.560.5.vhCDR3, CHA. 9.560. 5.vlCDR1, CHA. 9.560. 5.vlCDR2 and CHA. 9.560.5.vhCDR3;

CHA.9.560.6, CHA. 9.560. 6VH, CHA. 9.560. 6.VL, CHA. 9.560. 6.HC, CHA.9.560. 6.LC, CHA. 9.560. 6.H1, CHA. 9.560. 6.H2, CHA. 9.560. 6.H3;CHA.9.560.6.H4, CHA.9.560.6.H4(S241P), CHA. 9.560. 6.vhCDR1, CHA. 9.560.6.vhCDR2, CHA. 9.560. 6.vhCDR3, CHA. 9.560. 6.vlCDR1, CHA. 9.560.6.vlCDR2 and CHA. 9.560. 6.vhCDR3;

CHA.9.560.7, CHA. 9.560. 7VH, CHA. 9.560. 7.VL, CHA. 9.560. 7.HC, CHA.9.560. 7.LC, CHA. 9.560. 7.H1, CHA. 9.560. 7.H2, CHA. 9.560. 7.H3;CHA.9.560.7.H4; CHA.9.560.7.H4(S241P); CHA. 9.560. 7.vhCDR1, CHA. 9.560.7.vhCDR2, CHA. 9.560. 7.vhCDR3, CHA. 9.560. 7.vlCDR1, CHA. 9.560.7.vlCDR2 and CHA. 9.560. 7.vhCDR3;

CHA.9.560.8, CHA. 9.560. 8VH, CHA. 9.560. 8.VL, CHA. 9.560. 8.HC, CHA.9.560. 8.LC, CHA. 9.560. 8.H1, CHA. 9.560. 8.H2, CHA. 9.560. 8.H3;CHA.9.560.8.H4, CHA.9.560.8.H4(S241P); CHA. 9.560. 8.vhCDR1, CHA. 9.560.8.vhCDR2, CHA. 9.560. 8.vhCDR3, CHA. 9.560. 8.vlCDR1, CHA. 9.560.8.vlCDR2 and CHA. 9.560. 8.vhCDR3;

CHA.9.546.1, CHA. 9. 546.1VH, CHA. 9. 546.1.VL, CHA. 9. 546.1.HC, CHA.9. 546.1.LC, CHA. 9. 546.1.H1, CHA. 9. 546.1.H2, CHA. 9. 546.1.H3;CHA.9.546.1.H4, CHA.9.546.1.H4(S241P), CHA. 9. 546.1.vhCDR1, CHA. 9.546.1.vhCDR2, CHA. 9. 546.1.vhCDR3, CHA. 9. 546.1.vlCDR1, CHA. 9.546.1.vlCDR2 and CHA. 9. 546.1.vhCDR3;

CHA.9.547.1, CHA. 9. 547.1VH, CHA. 9. 547.1.VL, CHA. 9. 547.1.HC, CHA.9. 547.1.LC, CHA. 9. 547.1.H1, CHA. 9. 547.1.H2, CHA. 9. 547.1.H3;CHA.9.547.1.H4, CHA.9.547.1.H4(S241P), CHA. 9. 547.1.vhCDR1, CHA. 9.547.1.vhCDR2, CHA. 9. 547.1.vhCDR3, CHA. 9. 547.1.vlCDR1, CHA. 9.547.1.vlCDR2 and CHA. 9. 547.1.vhCDR3;

CHA.9.547.2, CHA. 9. 547. 2VH, CHA. 9. 547. 2.VL, CHA. 9. 547. 2.HC,CHA. 9. 547. 2.LC, CHA. 9. 547. 2.H1, CHA. 9. 547. 2.H2, CHA. 9. 547.2.H3; CHA.9.547.2.H4, CHA.9.547.2.H4(S241P), CHA. 9. 547. 2.vhCDR1, CHA.9. 547. 2.vhCDR2, CHA. 9. 547. 2.vhCDR3, CHA. 9. 547. 2.vlCDR1, CHA. 9.547. 2.vlCDR2 and CHA. 9. 547. 2.vhCDR3;

CHA.9.547.3, CHA. 9. 547. 3VH, CHA. 9. 547. 3.VL, CHA. 9. 547. 3.HC,CHA. 9. 547. 3.LC, CHA. 9. 547. 3.H1, CHA. 9. 547. 3.H2, CHA. 9. 547.3.H3; CHA.9.547.3.H4, CHA.9.547.3.H4(S241P), CHA. 9. 547. 3.vhCDR1, CHA.9.547. 3.vhCDR2, CHA. 9. 547. 3.vhCDR3, CHA. 9. 547. 3.vlCDR1, CHA. 9.547. 3.vlCDR2 and CHA. 9. 547. 3.vhCDR3;

CHA.9.547.4, CHA. 9. 547. 4VH, CHA. 9. 547. 4.VL, CHA. 9. 547. 4.HC,CHA. 9.547. 4.LC, CHA. 9. 547. 4.H1, CHA. 9. 547. 4.H2, CHA. 9. 547.4.H3; CHA.9.547.4.H4, CHA.9.547.4.H4(S241P), CHA. 9. 547. 4.vhCDR1, CHA.9. 547. 4.vhCDR2, CHA. 9. 547. 4.vhCDR3, CHA. 9. 547. 4.vlCDR1, CHA. 9.547. 4.vlCDR2 and CHA. 9. 547. 4.vhCDR3;

CHA.9.547.6, CHA. 9. 547. 6 VH, CHA. 9. 547. 6.VL, CHA. 9. 547. 6.HC,CHA. 9. 547. 6.LC, CHA. 9. 547. 6.H1, CHA. 9. 547. 6.H2, CHA. 9. 547.6.H3; CHA.9.547.6.H4, CHA.9.547.6.H4(S241P), CHA. 9. 547. 6.vhCDR1, CHA.9. 547. 6.vhCDR2, CHA. 9. 547. 6.vhCDR3, CHA. 9. 547. 6.vlCDR1, CHA. 9.547. 6.vlCDR2 and CHA. 9. 547. 6.vhCDR3;

CHA.9.547.7, CHA. 9. 547. 7VH, CHA. 9. 547. 7.VL, CHA. 9. 547. 7.HC,CHA. 9. 547. 7.LC, CHA. 9. 547. 7.H1, CHA. 9. 547. 7.H2, CHA. 9. 547.7.H3; CHA.9.547.7.H4, CHA.9.547.7.H4(S241P), CHA. 9. 547. 7.vhCDR1, CHA.9. 547. 7.vhCDR2, CHA. 9. 547. 7.vhCDR3, CHA. 9. 547. 7.vlCDR1, CHA. 9.547. 7.vlCDR2 and CHA. 9. 547. 7.vhCDR3;

CHA.9.547.8, CHA. 9. 547. 8VH, CHA. 9. 547. 8.VL, CHA. 9. 547. 8.HC,CHA.9.547.8.LC, CHA. 9. 547. 8.H1, CHA. 9. 547. 8.H2, CHA. 9. 547. 8.H3;CHA.9.547.8.H4, CHA.9.547.8.H4(S241P), CHA. 9. 547. 8.vhCDR1, CHA. 9.547. 8.vhCDR2, CHA. 9. 547. 8.vhCDR3, CHA. 9. 547. 8.vlCDR1, CHA. 9.547. 8.vlCDR2 and CHA. 9. 547. 8.vhCDR3;

CHA.9.547.9, CHA.9.547.9, CHA.9.547.9VH, CHA.9.547.9.VL, CHA.9.547.9.HC, CHA.9.547.9.LC, CHA.9.547.9.H1, CHA.9.547.9.H2,CHA.9.547.9.H3; CHA.9.547.9.H4, CHA.9.547.9.H4, CHA.9.547.9.H4(S241P),CHA.9.547.9.H4(S241P), CHA.9.547.9.vhCDR1, CHA.9.547.9.vhCDR2,CHA.9.547.9.vhCDR3, CHA.9.547.9.vlCDR1, CHA.9.547.9.vlCDR2 andCHA.9.547.9.vhCDR3;

CHA.9.547.13, CHA.9.547.13, CHA.9.547. 13VH, CHA.9. 547.13.VL, CHA.9.547.13.HC, CHA. 9.547.13.LC, CHA. 9.547.13.H1, CHA.9.547.13.H2, CHA.9.547.13.H3; CHA.9.547.13.H4, CHA.9.547.13.H4, CHA.9.547.13.H4(S241P),CHA.9.547.13.H4(S241P), CHA. 9. 547.13.vhCDR1, CHA.9.547.13.vhCDR2,CHA.9.547. 13.vhCDR3, CHA. 9. 547.13.vlCDR1, CHA. 9. 547.13.vlCDR2 andCHA. 9. 547. 13.vhCDR3;

CHA.9.541.1, CHA. 9. 541.1.VH, CHA. 9. 541.1.VL, CHA. 9. 541.1.HC, CHA.9. 541.1.LC, CHA. 9. 541.1.H1, CHA. 9. 541.1.H2, CHA. 9. 541.1.H3;CHA.9.541.1.H4, CHA.9.541.1.H4(S241P), CHA. 9. 541.1.vhCDR1, CHA. 9.541.1.vhCDR2, CHA. 9. 541.1.vhCDR3, CHA. 9. 541.1.vlCDR1, CHA. 9.541.1.vlCDR2 and CHA. 9.541.1.vhCDR3;

CHA.9.541.3, CHA. 9. 541. 3.VH, CHA. 9. 541. 3.VL, CHA. 9. 541. 3.HC,CHA. 9. 541. 3.LC, CHA. 9. 541. 3.H1, CHA. 9. 541. 3.H2, CHA. 9. 541.3.H3; CHA.9.541.3.H4, CHA.9.541.3.H4(S241P), CHA. 9. 541. 3.vhCDR1, CHA.9. 541. 3.vhCDR2, CHA. 9. 541. 3.vhCDR3, CHA. 9. 541. 3.vlCDR1, CHA. 9.541. 3.vlCDR2 and CHA. 9.541. 3.vhCDR3;

CHA.9.541.4, CHA. 9. 541.4.VH, CHA. 9. 541. 4.VL, CHA. 9. 541. 4.HC,CHA. 9. 541. 4.LC, CHA. 9. 541. 4.H1, CHA. 9. 541. 4.H2, CHA. 9. 541.4.H3; CHA.9.541.4.H4, CHA.9.541.4.H4(S241P), CHA. 9. 541. 4.vhCDR1, CHA.9. 541. 4.vhCDR2, CHA. 9. 541. 4.vhCDR3, CHA. 9. 541. 4.vlCDR1, CHA. 9.541. 4.vlCDR2 and CHA. 9.541. 4.vhCDR3;

CHA.9.541.5, CHA. 9. 541. 5.VH, CHA. 9. 541. 5.VL, CHA. 9. 541. 5.HC,CHA. 9. 541. 5.LC, CHA. 9. 541. 5.H1, CHA. 9. 541. 5.H2, CHA. 9. 541.5.H3; CHA.9.541.5.H4, CHA.9.541.5.H4(S241P), CHA. 9. 541. 5.vhCDR1, CHA.9. 541. 5.vhCDR2, CHA. 9. 541. 5.vhCDR3, CHA. 9. 541. 5.vlCDR1, CHA. 9.541. 5.vlCDR2 and CHA. 9.541. 5.vhCDR3;

CHA.9.541.6, CHA. 9. 541. 6.VH, CHA. 9. 541. 6.VL, CHA. 9. 541. 6.HC,CHA. 9. 541. 6.LC, CHA. 9. 541. 6.H1, CHA. 9. 541. 6.H2, CHA. 9.541.6.H3; CHA.9.541.6.H4, CHA.9.541.6.H4(S241P), CHA. 9. 541. 6.vhCDR1,CHA. 9. 541. 6.vhCDR2, CHA. 9. 541. 6.vhCDR3, CHA. 9. 541. 6.vlCDR1,CHA. 9. 541. 6.vlCDR2 and CHA. 9.541. 6.vhCDR3;

CHA.9.541.7, CHA. 9. 541. 7.VH, CHA. 9. 541. 7.VL, CHA. 9. 541. 7.HC,CHA. 9. 541. 7.LC, CHA. 9. 541. 7.H1, CHA. 9. 541. 7.H2, CHA. 9. 541.7.H3; CHA.9.541.7.H4, CHA.9.541.7.H4(S241P), CHA. 9. 541. 7.vhCDR1, CHA.9. 541. 7.vhCDR2, CHA. 9. 541. 7.vhCDR3, CHA. 9. 541. 7.vlCDR1, CHA. 9.541. 7.vlCDR2 and CHA. 9.541. 7.vhCDR3; and

CHA.9.541.8, CHA. 9. 541. 8.VH, CHA. 9. 541. 8.VL, CHA. 9. 541. 8.HC,CHA. 9. 541. 8.LC, CHA. 9. 541. 8.H1, CHA. 9. 541. 8.H2, CHA. 9. 541.8.H3; CHA.9.541.8.H4, CHA.9.541.8.H4(S241P); CHA. 9. 541. 8vhCDR1, CHA.9. 541. 8.vhCDR2, CHA. 9. 541. 8.vhCDR3, CHA. 9. 541. 8.vlCDR1, CHA. 9.541. 8.vlCDR2 and CHA. 9.541. 8.vhCDR3.

In the case of scFvs comprising the CDRs of the antibodies above, theseare labeled as scFvs that include a scFv comprising a variable heavydomain with the vhCDRs, a linker and a variable light domain with thevlCDRs, again as above in either orientation. Thus the inventionincludes the use of scFv-CHA.9.536.3.1, scFv-CHA.9.536.3,scFv-CHA.9.536.4, scFv-CHA.9.536.5, scFv-CHA.9.536.7, scFv-CHA.9.536.8,scFv-CHA.9.560.1, scFv-CHA.9.560.3, scFv-CHA.9.560.4, scFv-CHA.9.560.5,scFv-CHA.9.560.6, scFv-CHA.9.560.7, scFv-CHA.9.560.8, scFv-CHA.9.546.1,scFv-CHA.9.547.1, scFv-CHA.9.547.2, scFv-CHA.9.547.3, scFv-CHA.9.547.4,scFv-CHA.9.547.6, scFv-CHA.9.547.7, scFv-CHA.9.547.8, scFv-CHA.9.547.9,scFv-CHA.9.547.13, scFv-CHA.9.541.1, scFv-CHA.9.541.3, scFv-CHA.9.541.4,scFv-CHA.9.541.5, scFv-CHA.9.541.6, scFv-CHA.9.541.7 andscFv-CHA.9.541.8.

In addition, CHA.9.543 binds to TIGIT but does not block the TIGIT-PVRinteraction.

As discussed herein, the invention further provides for the use ofvariants of the above components (CPA and CHA), including variants inthe CDRs, as outlined above. Thus, the invention provides antibodiescomprising a set of 6 CDRs as outlined herein that can contain one, twoor three amino acid differences in the set of CDRs, as long as theantibody still binds to TIGIT. Suitable assays for testing whether ananti-TIGIT antibody that contains mutations as compared to the CDRsequences outlined herein are known in the art, such as Biacore assays.

In addition, the invention further provides for the use of variants ofthe above variable heavy and light chains. In this case, the variableheavy chains can be 80%, 90%, 95%, 98% or 99% identical to the “VH”sequences herein, and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10amino acid changes, or more, when Fc variants are used. Variable lightchains are provided that can be 80%, 90%, 95%, 98% or 99% identical tothe “VL” sequences herein (and in particular CPA.9.086), and/or containfrom 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid changes, or more, when Fcvariants are used. In these embodiments, the anti-TIGIT antibodies foruse in the present invention still binds to TIGIT. Suitable assays fortesting whether an anti-TIGIT antibody that contains mutations ascompared to the CDR sequences outlined herein are known in the art, suchas Biacore assays.

Similarly, heavy and light chains are provided that are 80%, 90%, 95%,98% or 99% identical to the full length “HC” and “LC” sequences herein(and in particular CPA.9.086), and/or contain from 1, 2, 3, 4, 5, 6, 7,8, 9, 10 amino acid changes, or more, when Fc variants are used. Inthese embodiments, the invention includes these variants as long as theanti-TIGIT antibody still binds to TIGIT. Suitable assays for testingwhether an anti-TIGIT antibody that contains mutations as compared tothe CDR sequences outlined herein are known in the art, such as Biacoreassays.

In addition, the framework regions of the variable heavy and variablelight chains of either the CPA or CHA antibodies herein can be humanized(or, in the case of the CHA antibodies, “rehumanized”, to the extentthat alternative humanization methods can be done) as is known in theart (with occasional variants generated in the CDRs as needed), and thushumanized variants of the VH and VL chains of FIG. 23 can be generated(and in particular CPA.9.086). Furthermore, the humanized variable heavyand light domains can then be fused with human constant regions, such asthe constant regions from IgG1, IgG2, IgG3 and IgG4 (includingIgG4(S241P)).

In particular, as is known in the art, murine VH and VL chains can behumanized as is known in the art, for example, using the IgBLAST programof the NCBI website, as outlined in Ye et al. Nucleic Acids Res.41:W34-W40 (2013), herein incorporated by reference in its entirety forthe humanization methods. IgBLAST takes a murine VH and/or VL sequenceand compares it to a library of known human germline sequences. As shownherein, for the humanized sequences generated herein, the databases usedwere IMGT human VH genes (F+ORF, 273 germline sequences) and IMGT humanVL kappa genes (F+ORF, 74 germline sequences). An exemplary five CHAsequences were chosen: CHA.9.536, CHA9.560, CHA.9.546, CHA.9.547 andCHA.9.541 (see FIG. 3 ). For this embodiment of the humanization, humangermline IGHV1-46(allelel) was chosen for all 5 as the acceptor sequenceand the human heavy chain IGHJ4(allelel) joining region (J gene). Forthree of four (CHA.7.518, CHA.7.530, CHA.7.538_1 and CHA.7.538_2), humangermline IGKV1-39(allele 1) was chosen as the acceptor sequence andhuman light chain IGKJ2(allelel) (J gene) was chosen. The J gene waschosen from human joining region sequences compiled at IMGT® theinternational ImMunoGeneTics information system as www.imgt.org. CDRswere defined according to the AbM definition (seewww.bioinfo.org.uk/abs/). In some embodiments, the anti-TIGIT antibodiesfor use in the present invention include TIGIT binding portions orantigen binding domains wherein the V_(H) and V_(L) sequences ofdifferent TIGIT binding portions or antigen binding domains can be“mixed and matched” to create other TIGIT binding portions or antigenbinding domains. TIGIT binding of such “mixed and matched” anti-TIGITantibodies can be tested using the binding assays described above. e.g.,ELISAs or Biacore assays). In some embodiments, when V_(H) and V_(L)chains are mixed and matched, a V_(H) sequence from a particularV_(H)/V_(L) pairing is replaced with a structurally similar V_(H)sequence. Likewise, in some embodiments, a V_(L) sequence from aparticular V_(H)/V_(L) pairing is replaced with a structurally similarV_(L) sequence. For example, the V_(H) and V_(L) sequences of homologousantibodies are particularly amenable for mixing and matching.

Accordingly, the anti-TIGIT antibodies for use in the present inventioncan comprise CDR amino acid sequences selected from the group consistingof (a) sequences as listed herein; (b) sequences that differ from thoseCDR amino acid sequences specified in (a) by 1, 2, 3, 4, 5, 6, 7, 8, 9,10 or more amino acid substitutions; (c) amino acid sequences having 90%or greater, 95% or greater, 98% or greater, or 99% or greater sequenceidentity to the sequences specified in (a) or (b); (d) a polypeptidehaving an amino acid sequence encoded by a polynucleotide having anucleic acid sequence encoding the amino acids as listed herein. Inparticular, the anti-TIGIT antibody can comprise the antigen bindingdomain from the CPA.9.086 antibody which can have sequences selectedfrom (a), (b), (c) or (d).

Additionally included in the definition of the anti-TIGIT antibodies foruse in the present invention are antibodies that comprise TIGIT bindingdomains that share identity to the binding domains from the TIGITantibodies enumerated herein. That is, in certain embodiments, ananti-TIGIT antibody according to the invention comprises heavy and lightchain variable regions comprising amino acid sequences that areidentical to all or part of the binding domains from the anti-TIGITamino acid sequences of preferred anti-TIGIT antibodies, respectively,wherein the antibodies retain the desired functional properties of theparent anti-TIGIT antibodies. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % homology=# of identical positions/total # ofpositions X 100), taking into account the number of gaps, and the lengthof each gap, which need to be introduced for optimal alignment of thetwo sequences. The comparison of sequences and determination of percentidentity between two sequences can be accomplished using a mathematicalalgorithm, as described in the non-limiting examples below.

The percent identity between two amino acid sequences can be determinedusing the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci.,4:11-17 (1988)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availablecommercially), using either a Blossum 62 matrix or a PAM250 matrix, anda gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2,3, 4, 5, or 6.

In general, the percentage identity for comparison between TIGIT bindingdomains or antigen binding domains is at least 75%, at least 80%, atleast 90%, with at least about 95%, 96%, 97%, 98% or 99% percentidentity being preferred. The percentage identity may be along the wholeamino acid sequence, for example the entire heavy or light chain oralong a portion of the chains. For example, included within thedefinition of the anti-TIGIT antibodies for use in the present inventionare those whose TIGIT binding portion or antigen binding domains sharesidentity along the entire variable region (for example, where theidentity is 95% or 98% identical along the variable regions), or alongthe entire constant region, or along just the Fc domain. In particular,the anti-TIGIT antibodies for use in the present invention includeantibodies that have TIGIT binding portions or antigen binding domainswith at least 75%, at least 80%, at least 90%, with at least about 95%,96%, 97%, 98%, or 99% percent identity being preferred, with theCPA.9.086 antibody.

In addition, also included are sequences that may have the identicalCDRs but changes in the framework portions of the variable domain (orentire heavy or light chain). For example, anti-TIGIT antibodies for usein the present invention include those with CDRs identical to thoseshown in FIG. 3 but whose identity along the variable region can belower, for example 95 or 98% percent identical. In particular, theinvention provides for the use of anti-TIGIT antibodies that have TIGITbinding portions or antigen binding domains with identical CDRs toCPA.9.086 but with framework regions that are 95% or 98% identical toCPA.9.086.

D. Anti-TIGIT Antibodies in Combination with anti-PD-1 Antibodies

In another embodiment, the invention provides combinations of theanti-TIGIT antibodies of the invention and anti-PD-1 antibodies. Thereare two approved anti-PD-1 antibodies, pembrolizumab (Keytruda®) andnivolumab (Opdivo®) and many more in development which can be used incombination with the anti-TIGIT antibodies of the invention.

Accordingly, the invention provides the specific combinations of:CPA.9.083.H4(S241P) as shown in FIG. 3F with pembrolizumab;CPA.9.083.H4(S241P) as shown in FIG. 3F with nivolumab;CPA.9.086.H4(S241P) as shown in FIG. 3G with pembrolizumab;CPA.9.086.H4(S241P) as shown in FIG. 3G with nivolumab;CHA.9.547.7H4(S241P) as shown in FIG. 4HH with pembrolizumab;CHA.9.547.7H4(S241P) as shown in FIG. 3HH with nivolumab;CHA.9.547.13.H4(S241P) as shown in FIG. 3VV with pembrolizumab andCHA.9.547.13.H4(S241P) as shown in FIG. 3VV with nivolumab; all fromFIG. 4 of U.S. Ser. No. 62/513,916, entitled “Anti-TIGIT Antibodies andMethods of Use”, filed on Jun. 1, 2017, by assignee Compugen. Otheranti-TIGIT antibodies that can be combined with anti-PD-1 antibodies areprovided in FIG. 3 as well.

E. Specific Anti-PVRIG Antibodies

The invention provides antigen binding domains, including full lengthantibodies, which contain a number of specific, enumerated sets of 6CDRs and defined variable heavy (vh, VH or V_(H)) and variable light(vl, VL or V_(L)), that bind to PVRIG.

In one embodiment, the anti- PVRIG antibody is an antibody comprising aset of six CDRs (vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2 and vlCDR3) fromCHA.7.518.1.H4(S241P) as depicted in FIG. 5 . In one embodiment, theanti-TIGIT antibody is an antibody comprising the variable heavy (vh)and variable light (vl) domains from CHA.7.518.1.H4(S241P) as depictedin FIG. 5 , linked to a human IgG constant domain of IgG1, IgG2, IgG3,IgG4 and IgG4(S241P). In one embodiment, the anti-TIGIT antibody isCHA.7.518.1.H4(S241P). In some embodiments, the anti-PVRIG antibody isan anti-PVRIG antibody as shown in FIG. 5 or FIG. 63 .

In particular, the 2H6 anti-PVRIG antibody of Zhu et al., WO2017/041004,specifically incorporated herein by reference, can be used, which has avhCDR1 of SEQ ID NO:6, a vhCDR2 of SEQ ID NO:7, a vhCDR3 of SEQ ID NO:8,a vlCDR1 of SEQ ID NO:9, a vlCDR2 of SEQ ID NO:10 and a vhCDR3 of SEQ IDNO:11 from WO2017/041004. The 2H6 anti-PVRIG antibody of Zhu et al. hasa variable heavy domain comprising SEQ ID NO:6 and a variable lightdomain comprising SEQ ID NO:3, which can be linked to a human IgGconstant domain of IgG1, IgG2, IgG3, IgG4 and IgG4(S241P). All SEQ IDsin this paragraph are from WO2017/041004 and are also provided in FIG. 5.

In particular, the 334M5 anti-PVRIG antibody from WO2018/017864,specifically incorporated herein by reference, can be used, which has avhCDR1 of SEQ ID NO:31, a vhCDR2 of SEQ ID NO:32, a vhCDR3 of SEQ IDNO:33, a vlCDR1 of SEQ ID NO:26, a vlCDR2 of SEQ ID NO:27, and a vhCDR3of SEQ ID NO:28 from WO2018/017864. The 334M5 anti-PVRIG antibody fromWO2018/017864 has a variable heavy domain comprising SEQ ID NO:30 and avariable light domain comprising SEQ ID NO:25, which can be linked to ahuman IgG constant domain of IgG1, IgG2, IgG3, IgG4 and IgG4(S241P). AllSEQ IDs in this paragraph are from WO2018/017864 and are also providedin FIG. 5 .

F. Additional Anti-PVRIG Antibodies

The PVRIG antibodies which can find use in according to the triplecombinations of the present invention are labeled as follows. ThesePVRIG antibodies described herein are labeled as follows. The PVRIGantibodies have reference numbers, for example “CPA.7.013”. Thisrepresents the combination of the variable heavy and variable lightchains, as depicted in FIG. 63 , for example. “CPA.7.013.VH” refers tothe variable heavy portion of CPA.7.013, while “CPA.7.013.VL” is thevariable light chain. “CPA.7.013.vhCDR1”, “CPA.7.013.vhCDR2”,“CPA.7.013.vhCDR3”, “CPA.7.013.vlCDR1”, “CPA.7.013.vlCDR2”, and“CPA.7.013.vlCDR3”, refers to the CDRs are indicated. “CPA.7.013.HC”refers to the entire heavy chain (e.g. variable and constant domain) ofthis molecule, and “CPA.7.013.LC” refers to the entire light chain (e.g.variable and constant domain) of the same molecule. “CPA.7.013.H1”refers to a full length antibody comprising the variable heavy and lightdomains, including the constant domain of Human IgG1 (hence, the H1;IgG1, IgG2, IgG3 and IgG4, as provided in FIG. 1 , for example).Accordingly, “CPA.7.013.H2” would be the CPA.7.013 variable domainslinked to a Human IgG2. “CPA.7.013.H3” would be the CPA.7.013 variabledomains linked to a Human IgG3, and “CPA.7.013.H4” would be theCPA.7.013 variable domains linked to a Human IgG4.

The PVRIG antibodies which can find use in according to the triplecombinations of the present invention are labeled as follows. Theantibodies have reference numbers, for example “CHA.7.518.1”. Thisrepresents the combination of the variable heavy and variable lightchains, as depicted in FIGS. 5 and 63 , for example, with theunderstanding that these antibodies include two heavy chains and twolight chains. “CPA.7.518.1.VH” refers to the variable heavy portion ofCPA.7.518.1, while “CPA.7.518.1.VL” is the variable light chain.“CPA.7.518.1.vhCDR1”, “CPA.7.518.1.vhCDR2”, “CPA.7.518.1.vhCDR3”,“CPA.7.518.1.vlCDR1”, “CPA.7.518.1.vlCDR2”, and “CPA.7.518.1.vlCDR3”,refers to the CDRs are indicated. “CPA.7.518.1.HC” refers to the entireheavy chain (e.g. variable and constant domain) of this molecule, and“CPA.7.518.1.LC” refers to the entire light chain (e.g. variable andconstant domain) of the same molecule. In general, the human kappa lightchain is used for the constant domain of each phage (or humanizedhybridoma) antibody herein, although in some embodiments the lambdalight constant domain is used. “CPA. 7.518.1.H1” refers to a full-lengthantibody comprising the variable heavy and light domains, including theconstant domain of Human IgG1 (hence, the H1; IgG1, IgG2, IgG3 and IgG4,as provided in FIG. 1 , for example). Accordingly, “CPA.7.518.1.H2”would be the CPA. 7.518.1 variable domains linked to a Human IgG2.“CPA.7.518.1.H3” would be the CPA. 7.518.1 variable domains linked to aHuman IgG3, and “CPA.7.518.1.H4” would be the CPA.7.518.1 variabledomains linked to a Human IgG4. Note that in some cases, the human IgGsmay have additional mutations, such are described below, and this can beannotated. For example, in many embodiments, there may be a S241Pmutation in the human IgG4, and this can be annotated as“CPA.7.518.1.H4(S241P)” for example. The human IgG4 sequence with thisS241P hinge variant is shown in FIG. 1 . Other potential variants areIgG1(N297A), (or other variants that ablate glycosylation at this siteand thus many of the effector functions associated with FcγRIIIabinding), and IgG1(D265A), which reduces binding to FcγR receptors. Theanti-PVRIG antibodies for use in the present invention can comprise anyof the PVRIG antibody sequences. The anti-PVRIG antibodies for use inthe present invention can comprise any of the PVRIG antigen bindingdomain sequences.

The invention further provides variable heavy and light domains as wellas full length heavy and light chains, any of which can be employed aspart of the anti-PVRIG antibodies for use according to the presentinvention.

In some embodiments, the invention provides scFvs that bind to PVRIGcomprising a variable heavy domain and a variable light domain linked byan scFv linker as outlined above. The VL and VH domains can be in eitherorientation, e.g. from N- to C-terminus “VH-linker-VL” or “VL-linker”VH″. These are named by their component parts; for example,“scFv-CHA.7.518.1VH-linker-VL” or “scFv-CPA.7.518.1. VL-linker-VH.”Thus, “scFv-CPA.7.518.1” can be in either orientation. The anti-PVRIGantibodies for use in the present invention can comprise an scFv thatbinds to PVRIG.

The invention provides antigen binding domains, including full lengthantibodies, which contain a number of specific, enumerated sets of 6CDRs. The anti-PVRIG antibodies for use in the present invention cancomprise any of the sets of 6 CDRs from the PVRIG antibody sequencesprovided herein.

The invention further provides variable heavy and light domains as wellas full length heavy and light chains.

In many embodiments, the anti-PVRIG antibodies for use in the presentinvention are human (derived from phage) and block binding of PVRIG andPVLR2. The anti-PVRIG antibodies of the invention can comprise a PVRIGantibody and/or antigen binding domain sequence capable of both bindingand blocking the receptor-ligand interaction. The anti-PVRIG cancomprise the CDRs from a PVRIG antibody sequence capable of both bindingand blocking the receptor-ligand interaction. The CPA antibodies, aswell as the CDR sequences, that both bind and block the receptor-ligandinteraction are as below, with their components outlined as well, thesequences for which are shown in FIG. 63 :

CPA.7.001, CPA.7.001.VH, CPA.7.001.VL, CPA.7.001.HC, CPA.7.001.LC andCPA.7.001.H1, CPA.7.001.H2, CPA.7.001.H3, CPA.7.001.H4;CPA.7.001.vhCDR1, CPA.7.001.vhCDR2, CPA.7.001.vhCDR3, CPA.7.001.vlCDR1,CPA.7.001.vlCDR2, and CPA.7.001.vlCDR3;

CPA.7.003, CPA.7.003.VH, CPA.7.003.VL, CPA.7.003.HC, CPA.7.003.LC,CPA.7.003.H1, CPA.7.003.H2, CPA.7.003.H3, CPA.7.003.H4;CPA.7.003.vhCDR1, CPA.7.003.vhCDR2, CPA.7.003.vhCDR3, CPA.7.003.vlCDR1,CPA.7.003.vlCDR2, and CPA.7.003.vlCDR3;

CPA.7.004, CPA.7.004.VH, CPA.7.004.VL, CPA.7.004.HC, CPA.7.004.LC,CPA.7.004.H1, CPA.7.004.H2, CPA.7.004.H3 CPA.7.004.H4; CPA.7.004.vhCDR1,CPA.7.004.vhCDR2, CPA.7.004.vhCDR3, CPA.7.004.vlCDR1, CPA.7.004.vlCDR2,and CPA.7.004.vlCDR3;

CPA.7.006, CPA.7.006.VH, CPA.7.006.VL, CPA.7.006.HC, CPA.7.006.LC,CPA.7.006.H1, CPA.7.006.H2, CPA.7.006.H3 CPA.7.006.H4; CPA.7.006.vhCDR1,CPA.7.006.vhCDR2, CPA.7.006.vhCDR3, CPA.7.006.vlCDR1, CPA.7.006.vlCDR2,and CPA.7.006.vlCDR3;

CPA.7.008, CPA.7.008.VH, CPA.7.008.VL, CPA.7.008.HC, CPA.7.008.LC,CPA.7.008.H1, CPA.7.008.H2, CPA.7.008.H3 CPA.7.008.H4; CPA.7.008.vhCDR1,CPA.7.008.vhCDR2, CPA.7.008.vhCDR3, CPA.7.008.vlCDR1, CPA.7.008.vlCDR2,and CPA.7.008.vlCDR3;

CPA.7.009, CPA.7.009.VH, CPA.7.009.VL, CPA.7.009.HC, CPA.7.009.LC,CPA.7.009.H1, CPA.7.009.H2, CPA.7.009.H3 CPA.7.009.H4; CPA.7.009.vhCDR1,CPA.7.009.vhCDR2, CPA.7.009.vhCDR3, CPA.7.009.vlCDR1, CPA.7.009.vlCDR2,and CPA.7.009.vlCDR3;

CPA.7.010, CPA.7.010.VH, CPA.7.010.VL, CPA.7.010.HC, CPA.7.010.LC,CPA.7.010.H1, CPA.7.010.H2, CPA.7.010.H3 CPA.7.010.H4; CPA.7.010.vhCDR1,CPA.7.010.vhCDR2, CPA.7.010.vhCDR3, CPA.7.010.vlCDR1, CPA.7.010.vlCDR2,and CPA.7.010.vlCDR3;

CPA.7.011, CPA.7.011.VH, CPA.7.011.VL, CPA.7.011.HC, CPA.7.011.LC,CPA.7.011.H1, CPA.7.011.H2, CPA.7.011.H3 CPA.7.011.H4; CPA.7.011.vhCDR1,CPA.7.011.vhCDR2, CPA.7.011.vhCDR3, CPA.7.011.vlCDR1, CPA.7.011.vlCDR2,and CPA.7.011.vlCDR3;

CPA.7.012, CPA.7.012.VH, CPA.7.012.VL, CPA.7.012.HC, CPA.7.012.LC,CPA.7.012.H1, CPA.7.012.H2, CPA.7.012.H3 CPA.7.012.H4; CPA.7.012.vhCDR1,CPA.7.012.vhCDR2, CPA.7.012.vhCDR3, CPA.7.012.vlCDR1, CPA.7.012.vlCDR2,and CPA.7.012.vlCDR3;

CPA.7.013, CPA.7.013.VH, CPA.7.013.VL, CPA.7.013.HC, CPA.7.013.LC,CPA.7.013.H1, CPA.7.013.H2, CPA.7.013.H3 CPA.7.013.H4; CPA.7.013.vhCDR1,CPA.7.013.vhCDR2, CPA.7.013.vhCDR3, CPA.7.013.vlCDR1, CPA.7.013.vlCDR2,and CPA.7.013.vlCDR3;

CPA.7.014, CPA.7.014.VH, CPA.7.014.VL, CPA.7.014.HC, CPA.7.014.LC,CPA.7.014.H1, CPA.7.014.H2, CPA.7.014.H3 CPA.7.014.H4; CPA.7.014.vhCDR1,CPA.7.014.vhCDR2, CPA.7.014.vhCDR3, CPA.7.014.vlCDR1, CPA.7.014.vlCDR2,and CPA.7.014.vlCDR3;

CPA.7.015, CPA.7.015.VH, CPA.7.015.VL, CPA.7.015.HC, CPA.7.015.LC,CPA.7.015.H1, CPA.7.015.H2, CPA.7.015.H3 CPA.7.015.H4; CPA.7.015.vhCDR1,CPA.7.015.vhCDR2, CPA.7.015.vhCDR3, CPA.7.015.vlCDR1, CPA.7.015.vlCDR2,and CPA.7.015.vlCDR3;

CPA.7.017, CPA.7.017.VH, CPA.7.017.VL, CPA.7.017.HC, CPA.7.017.LC,CPA.7.017H1, CPA.7.017.H2, CPA.7.017.H3 CPA.7.017.H4; CPA.7.017.vhCDR1,CPA.7.000171.vhCDR2, CPA.7.017.vhCDR3, CPA.7.017.vlCDR1,CPA.7.017.vlCDR2, and CPA.7.017.vlCDR3;

CPA.7.018, CPA.7.018.VH, CPA.7.018.VL, CPA.7.018.HC, CPA.7.018.LC,CPA.7.018.H1, CPA.7.018.H2, CPA.7.018.H3 CPA.7.018.H4; CPA.7.017.vhCDR1,CPA.7.017.vhCDR2, CPA.7.017.vhCDR3, CPA.7.017.vlCDR1, CPA.7.017.vlCDR2,and CPA.7.017.vlCDR3;

CPA.7.019, CPA.7.019.VH, CPA.7.019.VL, CPA.7.019.HC, CPA.7.019.LC,CPA.7.019.H1, CPA.7.019.H2, CPA.7.019.H3 CPA.7.019.H4; CPA.7.019.vhCDR1,CPA.7.019.vhCDR2, CPA.7.019.vhCDR3, CPA.7.019.vlCDR1, CPA.7.019.vlCDR2,and CPA.7.019.vlCDR3;

CPA.7.021, CPA.7.021.VH, CPA.7.021.VL, CPA.7.021.HC, CPA.7.021.LC,CPA.7.021.H1, CPA.7.021.H2, CPA.7.021.H3 CPA.7.021.H4; CPA.7.021.vhCDR1,CPA.7.021.vhCDR2, CPA.7.021.vhCDR3, CPA.7.021.vlCDR1, CPA.7.021.vlCDR2,and CPA.7.021.vlCDR3;

CPA.7.022, CPA.7.022.VH, CPA.7.022.VL, CPA.7.022.HC, CPA.7.022.LC,CPA.7.022.H1, CPA.7.022.H2, CPA.7.022.H3 CPA.7.022.H4; CPA.7.022.vhCDR1,CPA.7.022.vhCDR2, CPA.7.002201.vhCDR3, CPA.7.022.vlCDR1,CPA.7.022.vlCDR2, and CPA.7.022.vlCDR3;

CPA.7.023, CPA.7.023.VH, CPA.7.023.VL, CPA.7.023.HC, CPA.7.023.LC,CPA.7.023.H1, CPA.7.023.H2, CPA.7.023.H3 CPA.7.023.H4; CPA.7.023.vhCDR1,CPA.7.023.vhCDR2, CPA.7.023.vhCDR3, CPA.7.023.vlCDR1, CPA.7.023.vlCDR2,and CPA.7.023.vlCDR3;

CPA.7.024, CPA.7.024.VH, CPA.7.024.VL, CPA.7.024.HC, CPA.7.024.LC,CPA.7.024.H1, CPA.7.024.H2, CPA.7.024.H3 CPA.7.024.H4; CPA.7.024.vhCDR1,CPA.7.024.vhCDR2, CPA.7.024.vhCDR3, CPA.7.024.vlCDR1, CPA.7.024.vlCDR2,and CPA.7.024.vlCDR3;

CPA.7.033, CPA.7.033.VH, CPA.7.033.VL, CPA.7.033.HC, CPA.7.033.LC,CPA.7.033.H1, CPA.7.033.H2, CPA.7.033.H3 CPA.7.033.H4; CPA.7.033.vhCDR1,CPA.7.033.vhCDR2, CPA.7.033.vhCDR3, CPA.7.033.vlCDR1, CPA.7.033.vlCDR2,and CPA.7.033.vlCDR3;

CPA.7.034, CPA.7.034.VH, CPA.7.034.VL, CPA.7.034.HC, CPA.7.034.LC,CPA.7.034.H1, CPA.7.034.H2, CPA.7.034.H3 CPA.7.034.H4; CPA.7.034.vhCDR1,

CPA.7.034.vhCDR2, CPA.7.034.vhCDR3, CPA.7.034.vlCDR1, CPA.7.034.vlCDR2,and CPA.7.034.vlCDR3;

CPA.7.036, CPA.7.036.VH, CPA.7.036.VL, CPA.7.036.HC, CPA.7.036.LC,CPA.7.036.H1, CPA.7.036.H2, CPA.7.036.H3 CPA.7.036.H4; CPA.7.036.vhCDR1,CPA.7.036.vhCDR2, CPA.7.036.vhCDR3, CPA.7.036.vlCDR1, CPA.7.036.vlCDR2,and CPA.7.036.vlCDR3;

CPA.7.040, CPA.7.040.VH, CPA.7.040.VL, CPA.7.040.HC, CPA.7.040.LC,CPA.7.040.H1, CPA.7.040.H2, CPA.7.040.H3 and CPA.7.040.H4;CPA.7.040.vhCDR1, CPA.7.040.vhCDR2, CPA.7.040.vhCDR3, CPA.7.040.vlCDR1,CPA.7.040.vlCDR2, and CPA.7.040.vlCDR3;

CPA.7.046, CPA.7.046.VH, CPA.7.046.VL, CPA.7.046.HC, CPA.7.046.LC,CPA.7.046.H1, CPA.7.046.H2, CPA.7.046.H3 CPA.7.046.H4; CPA.7.046.vhCDR1,CPA.7.046.vhCDR2, CPA.7.046.vhCDR3, CPA.7.046.vlCDR1, CPA.7.046.vlCDR2,and CPA.7.046.vlCDR3;

CPA.7.047, CPA.7.047.VH, CPA.7.047.VL, CPA.7.047.HC, CPA.7.047.LC,CPA.7.047.H1, CPA.7.047.H2, CPA.7.047.H3 CPA.7.047.H4; CPA.7.047.vhCDR1,CPA.7.047.vhCDR2, CPA.7.047.vhCDR3, CPA.7.047.vlCDR1,CPA.7.004701.vlCDR2, and CPA.7.047.vlCDR3;

CPA.7.049, CPA.7.049.VH, CPA.7.049.VL, CPA.7.049.HC, CPA.7.049.LC,CPA.7.049.H1, CPA.7.049.H2, CPA.7.049.H3 CPA.7.049.H4; CPA.7.049.vhCDR1,CPA.7.049.vhCDR2, CPA.7.049.vhCDR3, CPA.7.049.vlCDR1, CPA.7.049.vlCDR2,and CPA.7.049.vlCDR3; and

CPA.7.050, CPA.7.050.VH, CPA.7.050.VL, CPA.7.050.HC, CPA.7.050.LC,CPA.7.050.H1, CPA.7.050.H2, CPA.7.050.H3 CPA.7.050.H4, CPA.7.050.vhCDR1,CPA.7.050.vhCDR2, CPA.7.050.vhCDR3, CPA.7.050.vlCDR1, CPA.7.050.vlCDR2,and CPA.7.050.vlCDR3.

In addition, there are a number of CPA antibodies generated herein thatbound to PVRIG but did not block the interaction of PVRIG and PVLR2. Theanti-PVRIG antibodies for use in the present invention can comprise aPVRIG antibody and/or antigen binding domain sequence capable of bindingbut not blocking the receptor-ligand interaction. The anti-PVRIG for usein the present invention can comprise the CDRs from a PVRIG antibodysequence capable of sequence capable of binding but not blocking thereceptor-ligand interaction. The CPA antibodies, as well as the CDRsequences, that bind but do not block the receptor-ligand interactionare as below, with their components outlined as well, the sequences forwhich are shown in FIG. 63 :

CPA.7.028, CPA.7.028.VH, CPA.7.028.VL, CPA.7.028.HC, CPA.7.028.LC,CPA.7.028.H1, CPA.7.028.H2, CPA.7.028.H3 and CPA.7.028.H4;CPA.7.028.vhCDR1, CPA.7.028.vhCDR2, CPA.7.028.vhCDR3, CPA.7.028.vlCDR1,CPA.7.028.vlCDR2, and CPA.7.028.vlCDR3.

CPA.7.030, CPA.7.030.VH, CPA.7.030.VL, CPA.7.030.HC, CPA.7.030.LC,CPA.7.030.H1, CPA.7.030.H2, CPA.7.030.H3 and CPA.7.030.H4;CPA.7.030.vhCDR1, CPA.7.030.vhCDR2, CPA.7.030.vhCDR3, CPA.7.030.vlCDR1,CPA.7.030.vlCDR2, and CPA.7.030.vlCDR3.

CPA.7.041, CPA.7.041.VH, CPA.7.041.VL, CPA.7.041.HC, CPA.7.041.LC,CPA.7.041.H1, CPA.7.041.H2, CPA.7.041.H3 and CPA.7.041.H4;CPA.7.041.vhCDR1, CPA.7.041.vhCDR2, CPA.7.041.vhCDR3, CPA.7.041.vlCDR1,CPA.7.041.vlCDR2, and CPA.7.041.vlCDR3.

CPA.7.016, CPA.7.016.VH, CPA.7.016.VL, CPA.7.016.HC, CPA.7.016.LC,CPA.7.016.H1, CPA.7.016.H2, CPA.7.016.H3 and CPA.7.016.H4;CPA.7.016.vhCDR1, CPA.7.016.vhCDR2, CPA.7.016.vhCDR3, CPA.7.016.vlCDR1,CPA.7.016.vlCDR2, and CPA.7.016.vlCDR3.

CPA.7.020, CPA.7.020.VH, CPA.7.020.VL, CPA.7.020.HC, CPA.7.020.LC,CPA.7.020.H1, CPA.7.020.H2, CPA.7.020.H3 and CPA.7.020.H4;CPA.7.020.vhCDR1, CPA.7.020.vhCDR2, CPA.7.020.vhCDR3, CPA.7.020.vlCDR1,CPA.7.020.vlCDR2, and CPA.7.020.vlCDR3.

CPA.7.038, CPA.7.038.VH, CPA.7.038.VL, CPA.7.038.HC, CPA.7.038.LC,CPA.7.038.H1, CPA.7.038.H2, CPA.7.038.H3 and CPA.7.038.H4;CPA.7.038.vhCDR1, CPA.7.038.vhCDR2, CPA.7.038.vhCDR3, CPA.7.038.vlCDR1,CPA.7.038.vlCDR2, and CPA.7.038.vlCDR3.

CPA.7.044, CPA.7.044.VH, CPA.7.044.VL, CPA.7.044.HC, CPA.7.044.LC,CPA.7.044.H1, CPA.7.044.H2, CPA.7.044.H3 and CPA.7.044.H4;CPA.7.044.vhCDR1, CPA.7.044.vhCDR2, CPA.7.044.vhCDR3, CPA.7.044.vlCDR1,CPA.7.044.vlCDR2, and CPA.7.044.vlCDR3.

CPA.7.045, CPA.7.045.VH, CPA.7.045.VL, CPA.7.045.HC, CPA.7.045.LC,CPA.7.045.H1, CPA.7.045.H2, CPA.7.045.H3 and CPA.7.045.H4;CPA.7.045.vhCDR1, CPA.7.045.vhCDR2, CPA.7.045.vhCDR3, CPA.7.045.vlCDR1,CPA.7.045.vlCDR2, and CPA.7.045.vlCDR3.

As discussed herein, the invention further provides variants of theabove components, including variants in the CDRs, as outlined above. Inaddition, variable heavy chains can be 80%, 90%, 95%, 98% or 99%identical to the “VH” sequences herein, and/or contain from 1, 2, 3, 4,5, 6, 7, 8, 9, 10 amino acid changes, or more, when Fc variants areused. Variable light chains are provided that can be 80%, 90%, 95%, 98%or 99% identical to the “VL” sequences herein, and/or contain from 1, 2,3, 4, 5, 6, 7, 8, 9, 10 amino acid changes, or more, when Fc variantsare used. Similarly, heavy and light chains are provided that are 80%,90%, 95%, 98% or 99% identical to the “HC” and “LC” sequences herein,and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid changes, ormore, when Fc variants are used. The anti-PVRIG for use in the presentinvention can comprise any of these PVRIG antibody and/or antigenbindgin domain sequences.

Furthermore, the present invention provides a number of CHA antibodies,which are murine antibodies generated from hybridomas. As is well knownthe art, the six CDRs are useful when put into either human frameworkvariable heavy and variable light regions or when the variable heavy andlight domains are humanized. See, for example, FIGS. 5 and 63 .

The anti-PVRIG for use in the present the invention can comprise any ofthe following CHA sets of CDRs from PVRIG antibody sequences.Accordingly, the present invention provides for the use of anti-PVRIGthat comprise the following CHA sets of CDRs, the sequences of which areshown in FIG. 5 and/or FIG. 63 :

CHA.7.502.vhCDR1, CHA.7.502.vhCDR2, CHA.7.502.vhCDR3, CHA.7.502.vlCDR1,CHA.7.502.vlCDR2, and CHA.7.502.vlCDR3.

CHA.7.503.vhCDR1, CHA.7.503.vhCDR2, CHA.7.503.vhCDR3, CHA.7.503.vlCDR1,CHA.7.503.vlCDR2, and CHA.7.503.vlCDR3.

CHA.7.506.vhCDR1, CHA.7.506.vhCDR2, CHA.7.506.vhCDR3, CHA.7.506.vlCDR1,CHA.7.506.vlCDR2, and CHA.7.506.vlCDR3.

CHA.7.508.vhCDR1, CHA.7.508.vhCDR2, CHA.7.508.vhCDR3, CHA.7.508.vlCDR1,CHA.7.508.vlCDR2, and CHA.7.508.vlCDR3.

CHA.7.510.vhCDR1, CHA.7.510.vhCDR2, CHA.7.510.vhCDR3, CHA.7.510.vlCDR1,CHA.7.510.vlCDR2, and CHA.7.510.vlCDR3.

CHA.7.512.vhCDR1, CHA.7.512.vhCDR2, CHA.7.512.vhCDR3, CHA.7.512.vlCDR1,CHA.7.512.vlCDR2, and CHA.7.512.vlCDR3.

CHA.7.514.vhCDR1, CHA.7.514.vhCDR2, CHA.7.514.vhCDR3, CHA.7.514.vlCDR1,CHA.7.514.vlCDR2, and CHA.7.514.vlCDR3.

CHA.7.516.vhCDR1, CHA.7.516.vhCDR2, CHA.7.516.vhCDR3, CHA.7.516.vlCDR1,CHA.7.516.vlCDR2, and CHA.7.516.vlCDR3.

CHA.7.518.vhCDR1, CHA.7.518.vhCDR2, CHA.7.518.vhCDR3, CHA.7.518.vlCDR1,CHA.7.518.vlCDR2, and CHA.7.518.vlCDR3.

CHA.7.520_1.vhCDR1, CHA.7.520_1.vhCDR2, CHA.7.520_1.vhCDR3,CHA.7.520_1.vlCDR1, CHA.7.520_1.vlCDR2, and CHA.7.520 1.vlCDR3.

CHA.7.520_2.vhCDR1, CHA.7.520_2.vhCDR2, CHA.7.520_2.vhCDR3,CHA.7.520_2.vlCDR1, CHA.7.520_2.vlCDR2, and CHA.7.520_2.vlCDR3.

CHA.7.522.vhCDR1, CHA.7.522.vhCDR2, CHA.7.522.vhCDR3, CHA.7.522.vlCDR1,CHA.7.522.vlCDR2, and CHA.7.522.vlCDR3.

CHA.7.524.vhCDR1, CHA.7.524.vhCDR2, CHA.7.524.vhCDR3, CHA.7.524.vlCDR1,CHA.7.524.vlCDR2, and CHA.7.524.vlCDR3.

CHA.7.526.vhCDR1, CHA.7.526.vhCDR2, CHA.7.526.vhCDR3, CHA.7.526.vlCDR1,CHA.7.526.vlCDR2, and CHA.7.526.vlCDR3.

CHA.7.527.vhCDR1, CHA.7.527.vhCDR2, CHA.7.527.vhCDR3, CHA.7.527.vlCDR1,CHA.7.527.vlCDR2, and CHA.7.527.vlCDR3.

CHA.7.528.vhCDR1, CHA.7.528.vhCDR2, CHA.7.528.vhCDR3, CHA.7.528.vlCDR1,CHA.7.528.vlCDR2, and CHA.7.528.vlCDR3.

CHA.7.530.vhCDR1, CHA.7.530.vhCDR2, CHA.7.530.vhCDR3, CHA.7.530.vlCDR1,CHA.7.530.vlCDR2, and CHA.7.530.vlCDR3.

CHA.7.534.vhCDR1, CHA.7.534.vhCDR2, CHA.7.534.vhCDR3, CHA.7.534.vlCDR1,CHA.7.534.vlCDR2, and CHA.7.534.vlCDR3.

CHA.7.535.vhCDR1, CHA.7.535.vhCDR2, CHA.7.535.vhCDR3, CHA.7.535.vlCDR1,CHA.7.535.vlCDR2, and CHA.7.535.vlCDR3.

CHA.7.537.vhCDR1, CHA.7.537.vhCDR2, CHA.7.537.vhCDR3, CHA.7.537.vlCDR1,CHA.7.537.vlCDR2, and CHA.7.537.vlCDR3.

CHA.7.538_1.vhCDR1, CHA.7.538_1.vhCDR2, CHA.7.538_1.vhCDR3,CHA.7.538_1.vlCDR1, CHA.7.538_1.vlCDR2, and CHA.7.538_1.vlCDR3.

CHA.7.538_2.vhCDR1, CHA.7.538_2.vhCDR2, CHA.7.538_2.vhCDR3,CHA.7.538_2.vlCDR1, CHA.7.538_2.vlCDR2, and CHA.7.5382.vlCDR3.

CHA.7.543.vhCDR1, CHA.7.543.vhCDR2, CHA.7.543.vhCDR3, CHA.7.543.vlCDR1,CHA.7.543.vlCDR2, and CHA.7.543.vlCDR3.

CHA.7.544.vhCDR1, CHA.7.544.vhCDR2, CHA.7.544.vhCDR3, CHA.7.544.vlCDR1,CHA.7.544.vlCDR2, and CHA.7.544.vlCDR3.

CHA.7.545.vhCDR1, CHA.7.545.vhCDR2, CHA.7.545.vhCDR3, CHA.7.545.vlCDR1,CHA.7.545.vlCDR2, and CHA.7.545.vlCDR3.

CHA.7.546.vhCDR1, CHA.7.546.vhCDR2, CHA.7.546.vhCDR3, CHA.7.546.vlCDR1,CHA.7.546.vlCDR2, and CHA.7.546.vlCDR3.

CHA.7.547.vhCDR1, CHA.7.547.vhCDR2, CHA.7.547.vhCDR3, CHA.7.547.vlCDR1,CHA.7.547.vlCDR2, and CHA.7.547.vlCDR3.

CHA.7.548.vhCDR1, CHA.7.548.vhCDR2, CHA.7.548.vhCDR3, CHA.7.548.vlCDR1,CHA.7.548.vlCDR2, and CHA.7.548.vlCDR3.

CHA.7.549.vhCDR1, CHA.7.549.vhCDR2, CHA.7.549.vhCDR3, CHA.7.549.vlCDR1,CHA.7.549.vlCDR2, and CHA.7.549.vlCDR3.

CHA.7.550.vhCDR1, CHA.7.550.vhCDR2, CHA.7.550.vhCDR3, CHA.7.550.vlCDR1,CHA.7.550.vlCDR2, and CHA.7.550.vlCDR3.

As above, these sets of CDRs may also be amino acid variants asdescribed above.

In addition, the framework regions of the variable heavy and variablelight chains can be humanized as is known in the art (with occasionalvariants generated in the CDRs as needed), and thus humanized variantsof the VH and VL chains of FIG. 63 can be generated. Furthermore, thehumanized variable heavy and light domains can then be fused with humanconstant regions, such as the constant regions from IgG1, IgG2, IgG3 andIgG4.

In particular, as is known in the art, murine VH and VL chains can behumanized as is known in the art, for example, using the IgBLAST programof the NCBI website, as outlined in Ye et al. Nucleic Acids Res.41:W34-W40 (2013), herein incorporated by reference in its entirety forthe humanization methods. IgBLAST takes a murine VH and/or VL sequenceand compares it to a library of known human germline sequences. As shownherein, for the humanized sequences generated herein, the databases usedwere IMGT human VH genes (F+ORF, 273 germline sequences) and IMGT humanVL kappa genes (F+ORF, 74 germline sequences). An exemplary five CHAsequences were chosen: CHA.7.518, CHA.7.530, CHA.7.538_1, CHA.7.538_2and CHA.7.524 (see FIGS. 5 and 63 for the VH and VL sequences). For thisembodiment of the humanization, human germline IGHV1-46(allele1) waschosen for all 5 as the acceptor sequence and the human heavy chainIGHJ4(allelel) joining region (J gene). For three of four (CHA.7.518,CHA.7.530, CHA.7.538_1 and CHA.7.538_2), human germlineIGKV1-39(allele 1) was chosen as the acceptor sequence and human lightchain IGKJ2(allelel) (J gene) was chosen. The J gene was chosen fromhuman joining region sequences compiled at IMGT® the internationalImMunoGeneTics information system as www.imgt.org. CDRs were definedaccording to the AbM definition (see www.bioinfo.org.uk/abs/). FIG. 63also depicts humanized sequences as well as some potential changes tooptimize binding to PVRIG. The anti-PVRIG antibodies for use in thepresent invention can comprise any of these humanized PVRIG antibody orantigen bindgin domain sequences.

Specific humanized antibodies of CHA antibodies include those shown inFIGS. 5 and 63 , for example. The anti-PVRIG for use in the presentinvention can comprise CHA PVRIG antibody sequences as shown in FIG. 5or 63 . As will be appreciated by those in the art, each humanizedvariable heavy (Humanized Heavy; HH) and variable light (HumanizedLight, HL) sequence can be combined with the constant regions of humanIgG1, IgG2, IgG3 and IgG4. That is, CHA.7.518.HH1 is the first humanizedvariable heavy chain, and CHA.7.518.HH1.1 is the full length heavychain, comprising the “HH1” humanized sequence with a IgG1 constantregion (CHA.7.518.HH1.2 is CHA.7.518.HH1 with IgG2, etc.).

In some embodiments, the anti-PVRIG antibodies for use in the presentinvention include anti-PVRIG antibodies wherein the V_(H) and V_(L)sequences of different anti-PVRIG antibodies can be “mixed and matched”to create other anti-PVRIG antibodies. PVRIG binding of such “mixed andmatched” antibodies can be tested using the binding assays describedabove. e.g., ELISAs). In some embodiments, when V_(H) and V_(L) chainsare mixed and matched, a V_(H) sequence from a particular V_(H)/V_(L)pairing is replaced with a structurally similar V_(H) sequence.Likewise, in some embodiments, a V_(L) sequence from a particularV_(H)/V_(L) pairing is replaced with a structurally similar V_(L)sequence. For example, the V_(H) and V_(L) sequences of homologousantibodies are particularly amenable for mixing and matching. Theanti-PVRIG for use in the present invention can comprise PVRIG V_(H) andV_(L) sequences from different anti-PVRIG antibodies that have been“mixed and matched”.

Accordingly, the antibodies of the invention comprise CDR amino acidsequences selected from the group consisting of (a) sequences as listedherein; (b) sequences that differ from those CDR amino acid sequencesspecified in (a) by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acidsubstitutions; (c) amino acid sequences having 90% or greater, 95% orgreater, 98% or greater, or 99% or greater sequence identity to thesequences specified in (a) or (b); (d) a polypeptide having an aminoacid sequence encoded by a polynucleotide having a nucleic acid sequenceencoding the amino acids as listed herein. The anti-PVRIG for use in thepresent invention can comprise PVRIG variant CDR sequences.

Additionally included in the definition of PVRIG antibodies areantibodies that share identity to the anti-PVRIG antibodies enumeratedherein. That is, in certain embodiments, an anti-PVRIG antibodyaccording to the invention comprises heavy and light chain variableregions comprising amino acid sequences that are homologous to isolatedanti-PVRIG amino acid sequences of preferred anti-PVRIG immunemolecules, respectively, wherein the antibodies retain the desiredfunctional properties of the parent anti-PVRIG antibodies. The percentidentity between the two sequences is a function of the number ofidentical positions shared by the sequences (e.g., % homology=# ofidentical positions/total # of positions X 100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below. The anti-PVRIG antibodies for use in thepresent the invention can comprise heavy and light chain variableregions comprising amino acid sequences that are homologous to isolatedanti-PVRIG amino acid sequences as described herein.

The percent identity between two amino acid sequences can be determinedusing the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci.,4:11-17 (1988)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availablecommercially), using either a Blossum 62 matrix or a PAM250 matrix, anda gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2,3, 4, 5, or 6.

In general, the percentage identity for comparison between PVRIGantibodies is at least 75%, at least 80%, at least 90%, with at leastabout 95%, 96%, 97%, 98%, or 99% percent identity being preferred. Thepercentage identity may be along the whole amino acid sequence, forexample the entire heavy or light chain or along a portion of thechains. For example, included within the definition of the anti-PVRIGantibodies of the invention are those that share identity along theentire variable region (for example, where the identity is 95% or 98%identical along the variable regions), or along the entire constantregion, or along just the Fc domain.

G. TIGIT Antibodies with Anti-Tumor Antibodies

In some embodiments, the anti-TIGIT antibodies of the invention areco-administered with antibodies that, unlike immuno-oncology/checkpointinhibitors that generally act on the immune system to increase apatient's native immune response, instead are directed against aspecific tumor target antigen (TTA). There are a wide number of anti-TTAantibodies either approved or in development that can be combined withthe present TIGIT antibodies. Currently approved antibodies, include,but are not limited to, cetuximab, panitumumab, nimotuzumab (all toEGFR), rituximab (CD20), trastuzumab and pertuzumab (HER2), alemtuzumab(CD52), bevacizumab (VEGF), ofatumumab (CD20), denosumab (RANK ligand),brentuximab (CD30), daratumumab (CD38), ibritumomab (CD20) andipilimumab (CTLA-4). Specific target oncology antibodies in clinicaltrials that can be combined with the anti-TIGIT antibodies hereininclude, but are not limited to, anti-CTLA4 mAbs, such as ipilimumab,tremelimumab (see, for Example U.S. Patent Publication No.2017/0306025); anti-PD-1 such as nivolumab BMS-936558/MDX-1106/ONO-4538, AMP224, CT-011, MK-3475, anti-PD-L1 antagonists suchas Atezolizumab (IMpower133), BMS-936559/ MDX-1105, MEDI4736,RG-7446/MPDL3280A, as well as those described in U.S. Patent PublicaitonNo. 2017/0281764); anti-LAG-3 such as IMP-321, anti-TIM-3, anti-BTLA,anti-B7-H4, anti-B7-H3, Anti-VISTA; agonistic antibodies targetingimmunostimulatory proteins, including anti-CD40 mAbs such as CP-870,893,lucatumumab, dacetuzumab; anti-CD137 mAbs such as BMS-663513 urelumab(anti-4-1BB; see, for example, U.S. Pat. Nos. 7,288,638 and 8,962,804,incorporated by reference herein in their entireties); PF-05082566utomilumab (see, for example, U.S. Pat. Nos. 8,821,867; 8,337,850; and9,468,678, as well as International Patent Application Publication No.WO 2012/032433, incorporated by reference herein in their entireties);anti-OX40 mAbs, such as anti-OX40 (see, for example, WO2006/029879 orWO2010096418, incorporated by reference herein in their entireties);anti-GITR mAbs such as TRX518 (see, for example, U.S. Pat. No.7,812,135, incorporated by reference herein in its entirety); anti-CD27mAbs, such as varlilumab CDX-1127 (see, for example, WO 2016/145085 andU.S. Patent Publication Nos. US 2011/0274685 and US 2012/0213771,incorporated by reference herein in their entireties) anti-ICOS mAbs(for example, MEDI-570, JTX-2011, and anti-TIM3 antibodies (see, forexample, WO 2013/006490 or U.S. Patent Publication No. US 2016/0257758,incorporated by reference herein in their entireties), as well asmonoclonal antibodies to prostate cancer, ovarian cancer, breast cancer,endometrial cancer, multiple myeloma, melanoma, lymphomas, lung cancersincluding small cell lung cancer, kidney cancer, colorectal cancer,pancreatic cancer, gastric cancer, brain cancer, (see generallywww.clinicaltrials.gov).

H. Specific Anti-PD-1 Antibodies

In another embodiment, the invention provides combinations of theanti-TIGIT antibodies of the invention and anti-PD-1 antibodies. Thereare two approved anti-PD-1 antibodies, pembrolizumab (Keytruda®;MK-3475-033), cemiplimab (REGN2810; see US20170174779), and nivolumab(Opdivo®; CheckMate078) and many more in development which can be usedin combination with the anti-TIGIT antibodies of the invention. In otherembodiments, the anti-PD-1 antibody can include, for example, SHR-1210(CTR20160175 and CTR20170090), SHR-1210 (CTR20170299 and CTR20170322),JS-001 (CTR20160274), IBI308 (CTR20160735), BGB-A317 (CTR20160872)and/or a PD-1 antibody as recited in U.S. Patent Publication No.2017/0081409. Exemplary anti-PD-1 anitbody sequences are shown in FIG. 7and any of these can be used with the combination therapy methodsdescribed herein.

In some embodiments, the anti-TIGIT antibodies of the invention arecombined with anti-PVRIG antibodies as described herein as well asanti-PD-1 antibodies, as described herein or other anti-PD-1 antibodiesknown in the art, as a triple combination therapy.

Specific Anti-PD-L1 Antibodies

In another embodiment, the invention provides combinations of theanti-TIGIT antibodies of the invention and anti-PD-L1 antibodies. Thereare three approved anti-PD-L1 antibodies, atezolizumab (TECENTRIQ®;MPDL3280A), avelumab (BAVENCIO®; MSB001071 8C), and Durvalumab(MEDI4736), as well as other anti-PD-L1 antibodies in development.Numerous anti-PD-L1 antibodies are available and many more indevelopment which can be used in combination with the anti-TIGITantibodies of the invention. In embodiments, the PD-L1 antibody is onedescribed in U.S. Patent Publication No. 2017/0281764 as well asInternational Patent Publication No. WO 2013/079174 (avelumab) and WO2010/077634 (or U.S. Patent Application No. 20160222117 or U.S. Pat. No.8,217,149; atezolizumab). In some embodiments, the PD-L1 antibodycomprises a heavy chain sequence of SEQ ID NO: 34 and a light chainsequence of SEQ ID NO: 36 (from US 2017/281764). In some embodiments,the PD-L1 antibody is atezolizumab (TECENTRIQ®; MPDL3280A; IMpower110).In some embodiments, the PD-L1 antibody is avelumab (BAVENCIO®;MSB001071 8C). In some embodiments, the PD-L1 antibody is durvalumab(MEDI4736). In some embodiments, the PD-L1 antibody includes, forexample, Atezolizumab (IMpower133), BMS-936559/MDX-1105, and/orRG-7446/MPDL3280A, and/or YW243.55.570, as well as any of those providedherein in FIG. 62 .

In some embodiments, the anti-TIGIT antibodies of the invention arecombined with anti-PVRIG antibodies as described herein as well asanti-PD-L1 antibodies, as described herein or other anti-PD-L1antibodies known in the art, as a triple combination therapy.

J. Optional Antibody Engineering

The antibodies of the invention can be modified, or engineered, to alterthe amino acid sequences by amino acid substitutions. As discussedherein, amino acid substitutions can be made to alter the affinity ofthe CDRs for the antigen (including both increasing and decreasingbinding), as well as to alter additional functional properties of theantibodies. For example, the antibodies may be engineered to includemodifications within the Fc region, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. Furthermore, an antibody according to at leastsome embodiments of the invention may be chemically modified (e.g., oneor more chemical moieties can be attached to the antibody) or bemodified to alter its glycosylation, again to alter one or morefunctional properties of the antibody. Such embodiments are describedfurther below. The numbering of residues in the Fc region is that of theEU index of Kabat.

In one embodiment, the hinge region of CH1 is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer et al. The number of cysteine residues in thehinge region of CH1 is altered to, for example, facilitate assembly ofthe light and heavy chains or to increase or decrease the stability ofthe antibody.

In still another embodiment, the antibody can be modified to abrogate invivo Fab arm exchange, in particular when IgG4 constant domains areused. Specifically, this process involves the exchange of IgG4half-molecules (one heavy chain plus one light chain) between other IgG4antibodies that effectively results in antibodies which are functionallymonovalent. Mutations to the hinge region and constant domains of theheavy chain can abrogate this exchange (see Aalberse, RC, Schuurman J.,2002, Immunology 105:9-19). As outlined herein, a mutation that findsparticular use in the present invention is the S241P in the context ofan IgG4 constant domain. IgG4 finds use in the present invention as ithas no significant effector function, and is thus used to block thereceptor-ligand binding without cell depletion.

In some embodiments, amino acid substitutions can be made in the Fcregion, in general for altering binding to FcγR receptors. By “Fc gammareceptor”, “FcγR” or “FcgammaR” as used herein is meant any member ofthe family of proteins that bind the IgG antibody Fc region and isencoded by an FcγR gene. In humans this family includes but is notlimited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb, and FcγRIc;FcγRII (CD32), including isoforms FcγRIIa (including allotypes H131 andR131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; andFcγRIII (CD16), including isoforms FcγRIIIa (including allotypes V158and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 andFcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, entirelyincorporated by reference), as well as any undiscovered human FcγRs orFcγR isoforms or allotypes. An FcγR may be from any organism, includingbut not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγRsinclude but are not limited to FcγRI (CD64), FcγRII (CD32), FcγRIII-1(CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγRsor FcγR isoforms or allotypes.

There are a number of useful Fc substitutions that can be made to alterbinding to one or more of the FcγR receptors. Substitutions that resultin increased binding as well as decreased binding can be useful. Forexample, it is known that increased binding to FcγRIIIa generallyresults in increased ADCC (antibody dependent cell-mediatedcytotoxicity; the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell. Similarly, decreasedbinding to FcγRIIb (an inhibitory receptor) can be beneficial as well insome circumstances. Amino acid substitutions that find use in thepresent invention include those listed in U.S. Ser. Nos. 11/124,620(particularly FIG. 41 ) and U.S. Pat. No. 6,737,056, both of which areexpressly incorporated herein by reference in their entirety andspecifically for the variants disclosed therein.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector functions of the antibody. For example, one or more amino acidsselected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and322 can be replaced with a different amino acid residue such that theantibody has an altered affinity for an effector ligand but retains theantigen-binding ability of the parent antibody. The effector ligand towhich affinity is altered can be, for example, an Fc receptor or the Clcomponent of complement. This approach is described in further detail inU.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another example, one or more amino acids selected from amino acidresidues 329, 331 and 322 can be replaced with a different amino acidresidue such that the antibody has altered C1q binding and/or reduced orabolished complement dependent cytotoxicity (CDC). This approach isdescribed in further detail in U.S. Pat. Nos. 6,194,551 by Idusogie etal.

In another example, one or more amino acid residues within amino acidpositions 231 and 239 are altered to thereby alter the ability of theantibody to fix complement. This approach is described further in PCTPublication WO 94/29351 by Bodmer et al.

In yet another example, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcy receptor by modifying one or more amino acids at the followingpositions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268,269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294,295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326,327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378,382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. Thisapproach is described further in PCT Publication WO 00/42072 by Presta.Moreover, the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII andFcRn have been mapped and variants with improved binding have beendescribed (see Shields, R. L. et al. (2001) J. Biol. Chem.276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334and 339 are shown to improve binding to FcγRIII. Additionally, thefollowing combination mutants are shown to improve FcγRIII binding:T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A.Furthermore, mutations such as M252Y/S254T/T256E or M428L/N434S improvebinding to FcRn and increase antibody circulation half-life (see Chan CA and Carter P J (2010) Nature Rev Immunol 10:301-316).

In addition, the antibodies of the invention are modified to increaseits biological half-life. Various approaches are possible. For example,one or more of the following mutations can be introduced: T252L, T254S,T256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively,to increase the biological half-life, the antibody can be altered withinthe CH1 or CL region to contain a salvage receptor binding epitope takenfrom two loops of a CH2 domain of an Fc region of an IgG, as describedin U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al. Additionalmutations to increase serum half-life are disclosed in U.S. Pat. Nos.8,883,973, 6,737,056 and 7,371,826 and include 428L, 434A, 434S, and428L/434S.

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycosylated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for antigen or reduceeffector function such as ADCC. Such carbohydrate modifications can beaccomplished by, for example, altering one or more sites ofglycosylation within the antibody sequence, for example N297. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site, with an alaninereplacement finding use in some embodiments.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies according to at least some embodiments of theinvention to thereby produce an antibody with altered glycosylation. Forexample, the cell lines Ms704, Ms705, and Ms709 lack thefucosyltransferase gene, FUT8 (a (1,6) fucosyltransferase), such thatantibodies expressed in the Ms704, Ms705, and Ms709 cell lines lackfucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8 celllines are created by the targeted disruption of the FUT8 gene inCHO/DG44 cells using two replacement vectors (see U.S. PatentPublication No. 20040110704 by Yamane et al. and Yamane-Ohnuki et al.(2004) Biotechnol Bioeng 87:614-22). As another example, EP 1,176,195 byHanai et al. describes a cell line with a functionally disrupted FUT8gene, which encodes a fucosyl transferase, such that antibodiesexpressed in such a cell line exhibit hypofucosylation by reducing oreliminating the a 1,6 bond-related enzyme. Hanai et al. also describecell lines which have a low enzyme activity for adding fucose to theN-acetylglucosamine that binds to the Fc region of the antibody or doesnot have the enzyme activity, for example the rat myeloma cell lineYB2/0 (ATCC CRL 1662). PCT Publication WO 03/035835 by Presta describesa variant CHO cell line, Lec13 cells, with reduced ability to attachfucose to Asn(297)-linked carbohydrates, also resulting inhypofucosylation of antibodies expressed in that host cell (see alsoShields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740). PCTPublication WO 99/54342 by Umana et al. describes cell lines engineeredto express glycoprotein-modifying glycosyl transferases (e.g.,β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et al. (1999) Nat. Biotech. 17:176-180).Alternatively, the fucose residues of the antibody may be cleaved offusing a fucosidase enzyme. For example, the fucosidase α-L-fucosidaseremoves fucosyl residues from antibodies (Tarentino, A. L. et al. (1975)Biochem. 14:5516-23).

Another modification of the antibodies herein that is contemplated bythe invention is PEGylation or the addition of other water solublemoieties, typically polymers, e.g., in order to enhance half-life. Anantibody can be PEGylated to, for example, increase the biological(e.g., serum) half-life of the antibody. To PEGylate an antibody, theantibody, or fragment thereof, typically is reacted with polyethyleneglycol (PEG), such as a reactive ester or aldehyde derivative of PEG,under conditions in which one or more PEG groups become attached to theantibody or antibody fragment. Preferably, the PEGylation is carried outvia an acylation reaction or an alkylation reaction with a reactive PEGmolecule (or an analogous reactive water-soluble polymer). As usedherein, the term “polyethylene glycol” is intended to encompass any ofthe forms of PEG that have been used to derivatize other proteins, suchas mono (C₁-C₁₀) alkoxy- or aryloxy-polyethylene glycol or polyethyleneglycol-maleimide. In certain embodiments, the antibody to be PEGylatedis an aglycosylated antibody. Methods for PEGylating proteins are knownin the art and can be applied to the antibodies according to at leastsome embodiments of the invention. See for example, EP 0 154 316 byNishimura et al. and EP 0 401 384 by Ishikawa et al.

In addition to substitutions made to alter binding affinity to FcγRsand/or FcRn and/or increase in vivo serum half-life, additional antibodymodifications can be made, as described in further detail below.

In some cases, affinity maturation is done. Amino acid modifications inthe CDRs are sometimes referred to as “affinity maturation”. An“affinity matured” antibody is one having one or more alteration(s) inone or more CDRs which results in an improvement in the affinity of theantibody for antigen, compared to a parent antibody which does notpossess those alteration(s). In some cases, it may be desirable todecrease the affinity of an antibody to its antigen.

In some embodiments, one or more amino acid modifications are made inone or more of the CDRs of the antibodies of the invention. In general,only 1 or 2 or 3-amino acids are substituted in any single CDR, andgenerally no more than from 1, 2, 3. 4, 5, 6, 7, 8 9 or 10 changes aremade within a set of CDRs. However, it should be appreciated that anycombination of no substitutions, 1, 2 or 3 substitutions in any CDR canbe independently and optionally combined with any other substitution.

Affinity maturation can be done to increase the binding affinity of theantibody for the antigen by at least about 10% to 50-100-150% or more,or from 1 to 5 fold as compared to the “parent” antibody. Preferredaffinity matured antibodies will have nanomolar or even picomolaraffinities for the antigen. Affinity matured antibodies are produced byknown procedures. See, for example, Marks et al., 1992, Biotechnology10:779-783 that describes affinity maturation by variable heavy chain(VH) and variable light chain (VL) domain shuffling. Random mutagenesisof CDR and/or framework residues is described in: Barbas, et al. 1994,Proc. Nat. Acad. Sci, USA 91:3809-3813; Shier et al., 1995, Gene169:147-155; Yelton et al., 1995, J. Immunol. 155:1994-2004; Jackson etal., 1995, J. Immunol. 154(7):3310-9; and Hawkins et al, 1992, J. Mol.Biol. 226:889-896, for example.

Alternatively, amino acid modifications can be made in one or more ofthe CDRs of the antibodies of the invention that are “silent”, e.g. thatdo not significantly alter the affinity of the antibody for the antigen.These can be made for a number of reasons, including optimizingexpression (as can be done for the nucleic acids encoding the antibodiesof the invention).

Thus, included within the definition of the CDRs and antibodies of theinvention are variant CDRs and antibodies; that is, the antibodies ofthe invention can include amino acid modifications in one or more of theCDRs of the enumerated antibodies of the invention. In addition, asoutlined below, amino acid modifications can also independently andoptionally be made in any region outside the CDRs, including frameworkand constant regions.

VI. ANTI-TIGIT ANTIBODIES IN COMBINATION THERAPY

The TIGIT and PVRIG antibodies of the invention find particular use inthe treatment of cancer when used in combination and for example with acheckpoint inhibitor such as an anti-PD-1 antibody, as described herein.In general, the antibodies of the invention are immunomodulatory, inthat rather than directly attack cancerous cells, the anti-TIGIT andanti-PVRIG antibodies of the invention stimulate the immune system,generally by inhibiting the action of TIGIT and PVRIG, respectively.Thus, unlike tumor-targeted therapies, which are aimed at inhibitingmolecular pathways that are crucial for tumor growth and development,and/or depleting tumor cells, cancer immunotherapy is aimed to stimulatethe patient's own immune system to eliminate cancer cells, providinglong-lived tumor destruction. Various approaches can be used in cancerimmunotherapy, among them are therapeutic cancer vaccines to inducetumor-specific T cell responses, and immunostimulatory antibodies (i.e.antagonists of inhibitory receptors =immune checkpoints) to removeimmunosuppressive pathways.

Clinical responses with targeted therapy or conventional anti-cancertherapies tend to be transient as cancer cells develop resistance, andtumor recurrence takes place. However, the clinical use of cancerimmunotherapy in the past few years has shown that this type of therapycan have durable clinical responses, showing dramatic impact on longterm survival. However, although responses are long term, only a smallnumber of patients respond (as opposed to conventional or targetedtherapy, where a large number of patients respond, but responses aretransient).

By the time a tumor is detected clinically, it has already evaded theimmune-defense system by acquiring immunoresistant and immunosuppressiveproperties and creating an immunosuppressive tumor microenvironmentthrough various mechanisms and a variety of immune cells.

Accordingly, the anti-TIGIT and anti-PVRIG combinations of the inventionare useful in treating cancer. Due to the nature of an immuno-oncologymechanism of action, TIGIT and or PVRIG do not necessarily need to beoverexpressed on or correlated with a particular cancer type; that is,the goal is to have the anti-TIGIT antibodies de-suppress T cell and NKcell activation, such that the immune system will go after the cancers.

VII. NUCLEIC ACID COMPOSITIONS

Nucleic acid compositions encoding the anti-TIGIT, anti-PVRIG andanti-PD-1 antibodies of the invention are also provided, as well asexpression vectors containing the nucleic acids and host cellstransformed with the nucleic acid and/or expression vector compositions.As will be appreciated by those in the art, the protein sequencesdepicted herein can be encoded by any number of possible nucleic acidsequences, due to the degeneracy of the genetic code.

The nucleic acid compositions that encode the antibodies will depend onthe format of the antibody. For traditional, tetrameric antibodiescontaining two heavy chains and two light chains are encoded by twodifferent nucleic acids, one encoding the heavy chain and one encodingthe light chain. These can be put into a single expression vector or twoexpression vectors, as is known in the art, transformed into host cells,where they are expressed to form the antibodies of the invention. Insome embodiments, for example when scFv constructs are used, a singlenucleic acid encoding the variable heavy chain-linker-variable lightchain is generally used, which can be inserted into an expression vectorfor transformation into host cells. The nucleic acids can be put intoexpression vectors that contain the appropriate transcriptional andtranslational control sequences, including, but not limited to, signaland secretion sequences, regulatory sequences, promoters, origins ofreplication, selection genes, etc.

Preferred mammalian host cells for expressing the recombinant antibodiesaccording to at least some embodiments of the invention include ChineseHamster Ovary (CHO cells), PER.C6, HEK293 and others as is known in theart.

The nucleic acids may be present in whole cells, in a cell lysate, or ina partially purified or substantially pure form. A nucleic acid is“isolated” or “rendered substantially pure” when purified away fromother cellular components or other contaminants, e.g., other cellularnucleic acids or proteins, by standard techniques, includingalkaline/SDS treatment, CsCl banding, column chromatography, agarose gelelectrophoresis and others well known in the art.

To create a scFv gene, the VH- and VL-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly4-Ser)3, such that the VH and VLsequences can be expressed as a contiguous single-chain protein, withthe VL and VH regions joined by the flexible linker (see e.g., Bird etal. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad.Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

VIII. FORMULATIONS OF THE ANTIBODIES OF THE INVENTION

The therapeutic compositions used in the practice of the foregoingmethods can be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude any material that when combined with the therapeutic compositionretains the anti-tumor function of the therapeutic composition and isgenerally non-reactive with the patient's immune system. Examplesinclude, but are not limited to, any of a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like (see, generally,Remington's Pharmaceutical Sciences 16th Edition, A. Osal., Ed., 1980).Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed and may includebuffers.

In a preferred embodiment, the pharmaceutical composition that comprisesthe antibodies of the invention may be in a water-soluble form, such asbeing present as pharmaceutically acceptable salts, which is meant toinclude both acid and base addition salts. “Pharmaceutically acceptableacid addition salt” refers to those salts that retain the biologicaleffectiveness of the free bases and that are not biologically orotherwise undesirable, formed with inorganic acids and the like.“Pharmaceutically acceptable base addition salts” include those derivedfrom inorganic bases and the like.

Administration of the pharmaceutical composition comprising antibodiesof the present invention, preferably in the form of a sterile aqueoussolution, may be done in a variety of ways, including, but not limitedto subcutaneously and intravenously.

The dosing amounts and frequencies of administration are, in a preferredembodiment, selected to be therapeutically or prophylacticallyeffective. As is known in the art, adjustments for protein degradation,systemic versus localized delivery, and rate of new protease synthesis,as well as the age, body weight, general health, sex, diet, time ofadministration, drug interaction and the severity of the condition maybe necessary, and will be ascertainable with routine experimentation bythose skilled in the art.

In order to treat a patient, a therapeutically effective dose of the Fcvariant of the present invention may be administered. By“therapeutically effective dose” herein is meant a dose that producesthe effects for which it is administered. The exact dose will depend onthe purpose of the treatment, and will be ascertainable by one skilledin the art using known techniques.

A. Combination Formulations

The antibodies of the invention (either as a triple combination ofanti-TIGIT, anti-PVRIG and anti-PD-1 antibodies or as a doublecombination of anti-TIGIT and anti-PVRIG antibodies) can be done in avariety of ways as those in the art will appreciate. In some cases, theantibodies are administered simultaneously, either as separate infusions(e.g. each IV bag holds a single antibody), for example, or as oneinfusion of a mixture of the antibodies. Alternatively, the antibodiescan be administered sequentially, for example over a period of hours ordays.

In some cases, the antibodies are provided in an administration kit,with dosage units of each antibody, again either packaged separately inindividual dosage units, or together, as a mixture of antibodies as asingle dosage unit.

IX. COMBINATION THERAPIES AND USES

A. Cancer Therapies

“Cancer,” as used herein, refers broadly to any neoplastic disease(whether invasive or metastatic) characterized by abnormal anduncontrolled cell division causing malignant growth or tumor (e.g.,unregulated cell growth.) The term “cancer” or “cancerous” as usedherein should be understood to encompass any neoplastic disease (whetherinvasive, non-invasive or metastatic) which is characterized by abnormaland uncontrolled cell division causing malignant growth or tumor,non-limiting examples of which are described herein. This includes anyphysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer are exemplified in theworking examples and also are described within the specification.

Non-limiting examples of cancer that can be treated using anti-TIGITantibodies, anit-PVRIG antibodies, as well as combinations of anti-TIGITantibodies and other antibodies, such as any of the anti-TIGIT,anit-PVRIG, anti-PD-1 and/or anti-PD-L1 antibodies as provided herein.Such cancers include, but are not limited to, carcinoma, lymphoma,blastoma, sarcoma, and leukemia. More particular examples of suchcancers include squamous cell cancer, lung cancer (including small-celllung cancer, non-small cell lung cancer, adenocarcinoma of the lung, andsquamous carcinoma of the lung), cancer of the peritoneum,hepatocellular cancer, gastric or stomach cancer (includinggastrointestinal cancer), esophageal cancer, melanoma, mesothelioma,merkel cell cancer, pancreatic cancer, glioblastoma, cervical cancer,ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer,colon cancer, colorectal cancer, endometrial or uterine carcinoma,salivary gland carcinoma, kidney or renal cancer, prostate cancer,vulval cancer, thyroid cancer, hepatic carcinoma and various types ofhead and neck cancer, larynx cancer, oral cavity cancer, urothelialcancer, KRAS mutant tumors, Myelodysplastic syndromes (MDS), as well asB-cell malignancies, B-cell lymphoma (including low grade/follicularnon-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediategrade/follicular NHL; intermediate grade diffuse NHL; high gradeimmunoblastic NHL; high grade lymphoblastic NHL; high grade smallnon-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chroniclymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairycell leukemia; chronic myeloblastic leukemia; adult T-cellleukemia/lymphoma; myeloma; multiple myeloma and post-transplantlymphoproliferative disorder (PTLD), lymphoid malignancies, abnormalvascular proliferation associated with phakomatoses, edema (such as thatassociated with brain tumors), and Meigs' syndrome, rectal cancer, renalcell cancer, soft-tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma,ovarian early or advanced (including metastatic). The cancerousconditions amenable for treatment of the invention include cancers thatexpress or do not express TIGIT, PVRIG, PVRL, PD-1, and/or PD-L1, andfurther include non-metastatic or non-invasive as well as invasive ormetastatic cancers wherein TIGIT, PVRIG, PVRL, PD-1, and/or PD-L1,expression by immune, stromal or diseased cells suppress antitumorresponses and anti-invasive immune responses. The method of the presentinvention is particularly suitable for the treatment of vascularizedtumors. In some embodiments, the cancer is selected from the groupconsisting of prostate cancer, liver cancer (HCC), colorectal cancer,ovarian cancer, endometrial cancer, breast cancer, triple negativebreast cancer, pancreatic cancer, stomach (gastric) cancer, cervicalcancer, head and neck cancer, thyroid cancer, testis cancer, urothelialcancer, lung cancer (small cell lung, non-small cell lung), melanoma,non melanoma skin cancer (squamous and basal cell carcinoma), glioma,renal cancer (RCC), lymphoma (NHL or HL), Acute myeloid leukemia (AML),T cell Acute Lymphoblastic Leukemia (T-ALL), Diffuse Large B celllymphoma, testicular germ cell tumors, mesothelioma, esophageal cancer,Merkel Cells cancer, MSI-high cancer, KRAS mutant tumors, adult T-cellleukemia/lymphoma, and Myelodysplastic syndromes (MDS). In someembodiments of the method, the cancer is selected from the groupconsisting of cancer triple negative breast cancer, stomach (gastric)cancer, lung cancer (small cell lung, non-small cell lung), Merkel Cellscancer, MSI-high cancer, KRAS mutant tumors, adult T-cellleukemia/lymphoma, and Myelodysplastic syndromes (MDS).

“Cancer therapy” herein refers to any method which prevents or treatscancer or ameliorates one or more of the symptoms of cancer. Typically,such therapies will comprises administration of immunostimulatoryanti-TIGIT and anti-PVRIG antibodies (including antigen-bindingfragments) in combination with chemotherapy or radiotherapy or otherbiologics and for enhancing the activity thereof, i.e., in individualswherein expression of TIGIT and or PVRIG suppresses antitumor responsesand the efficacy of chemotherapy or radiotherapy or biologic efficacy.

The present invention provides combination therapies and uses ofanti-TIGIT antibodies and anti-PVRIG antibodies, sometimes with theaddition of anti-PD-1 antibodies, for a triple combination therapy. Thepresent invention provides combination therapies and uses of anti-TIGITantibodies and anti-PVRIG antibodies, sometimes with the addition ofanti-PD-L1 antibodies. Any of the PVRIG antibodies listed above or inthe figures can employed for a triple combination therapy. Any of theTIGIT antibodies listed above or in the figures can be employed for atriple combination therapy. Any of the PD-1 antibodies listed above orin the figures can employed for a triple combination therapy. Any of thePD-L1 antibodies listed above or in the figures can employed for atriple combination therapy. In some embodiments, the anti-TIGIT antibodyis an antibody chosen from any anti-TIGIT antibody described herein,including any of those described in FIG. 3 . In some embodiments, theanti-PVRIG antibody is an antibody chosen from any anti-PVRIG antibodydescribed herein, including any of those described in FIG. 5 and/or FIG.63 . In some embodiments, the anti-PD-1 antibody is an antibody chosenfrom any anti-PD-1 antibody described herein, including any of thosedescribed in FIG. 7 .

In some embodiments, the anti-PD-1 antibody is selected frompembrolizumab (Keytruda®; MK-3475-033), nivolumab (Opdivo®;CheckMate078), cemplimab (REGN2810), SHR-1210 (CTR20160175 andCTR20170090), SHR-1210 (CTR20170299 and CTR20170322), JS-001(CTR20160274), IBI308 (CTR20160735), BGB-A317 (CTR20160872) and/or aPD-1 antibody as recited in U.S. Patent Publication No. 2017/0081409.

In some embodiments, the anti-PD-L1 antibody is selected from antibodyis one described in U.S. Patent Publication No. 2017/0281764 as well asInternational Patent Publication No. WO 2013/079174 (avelumab) and WO2010/077634 (or U.S. Patent Application No. 20160222117 or U.S. Pat. No.8,217,149; atezolizumab). In some embodiments, the PD-L1 antibodycomprises a heavy chain sequence of SEQ ID NO: 34 and a light chainsequence of SEQ ID NO: 36 (from US 2017/281764). In some embodiments,the PD-L1 antibody is atezolizumab (TECENTRIQ®; MPDL3280A; IMpower110).In some embodiments, the PD-L1 antibody is avelumab (BAVENCIO®;MSB001071 8C). In some embodiments, the PD-L1 antibody is durvalumab(MEDI4736). In some embodiments, the PD-L1 antibody includes, forexample, Atezolizumab (IMpower133), BMS-936559/MDX-1105, and/orRG-7446/MPDL3280A, and/or YW243.55.S70, as well as any of those providedherein in FIG. 62 .

In some embodiments, the anti-PVRIG antibody is selected from anantibody the sequences for which are shown in FIG. 5 and/or 63:

-   CPA.7.001, CPA.7.001.VH, CPA.7.001.VL, CPA.7.001.HC, CPA.7.001.LC    and CPA.7.001.H1, CPA.7.001.H2, CPA.7.001.H3, CPA.7.001.H4;    CPA.7.001.vhCDR1, CPA.7.001.vhCDR2, CPA.7.001.vhCDR3,    CPA.7.001.vlCDR1, CPA.7.001.vlCDR2, and CPA.7.001.vlCDR3;-   CPA.7.003, CPA.7.003.VH, CPA.7.003.VL, CPA.7.003.HC, CPA.7.003.LC,    CPA.7.003.H1, CPA.7.003.H2, CPA.7.003.H3, CPA.7.003.H4;    CPA.7.003.vhCDR1, CPA.7.003.vhCDR2, CPA.7.003.vhCDR3,    CPA.7.003.vlCDR1, CPA.7.003.vlCDR2, and CPA.7.003.vlCDR3;-   CPA.7.004, CPA.7.004.VH, CPA.7.004.VL, CPA.7.004.HC, CPA.7.004.LC,    CPA.7.004.H1, CPA.7.004.H2, CPA.7.004.H3 CPA.7.004.H4;    CPA.7.004.vhCDR1, CPA.7.004.vhCDR2, CPA.7.004.vhCDR3,    CPA.7.004.vlCDR1, CPA.7.004.vlCDR2, and CPA.7.004.vlCDR3;-   CPA.7.006, CPA.7.006.VH, CPA.7.006.VL, CPA.7.006.HC, CPA.7.006.LC,    CPA.7.006.H1, CPA.7.006.H2, CPA.7.006.H3 CPA.7.006.H4;    CPA.7.006.vhCDR1, CPA.7.006.vhCDR2, CPA.7.006.vhCDR3,    CPA.7.006.vlCDR1, CPA.7.006.vlCDR2, and CPA.7.006.vlCDR3;-   CPA.7.008, CPA.7.008.VH, CPA.7.008.VL, CPA.7.008.HC, CPA.7.008.LC,    CPA.7.008.H1, CPA.7.008.H2, CPA.7.008.H3 CPA.7.008.H4;    CPA.7.008.vhCDR1, CPA.7.008.vhCDR2, CPA.7.008.vhCDR3,    CPA.7.008.vlCDR1, CPA.7.008.vlCDR2, and CPA.7.008.vlCDR3;-   CPA.7.009, CPA.7.009.VH, CPA.7.009.VL, CPA.7.009.HC, CPA.7.009.LC,    CPA.7.009.H1, CPA.7.009.H2, CPA.7.009.H3 CPA.7.009.H4;    CPA.7.009.vhCDR1, CPA.7.009.vhCDR2, CPA.7.009.vhCDR3,    CPA.7.009.vlCDR1, CPA.7.009.vlCDR2, and CPA.7.009.vlCDR3;-   CPA.7.010, CPA.7.010.VH, CPA.7.010.VL, CPA.7.010.HC, CPA.7.010.LC,    CPA.7.010.H1, CPA.7.010.H2, CPA.7.010.H3 CPA.7.010.H4;    CPA.7.010.vhCDR1, CPA.7.010.vhCDR2, CPA.7.010.vhCDR3,    CPA.7.010.vlCDR1, CPA.7.010.vlCDR2, and CPA.7.010.vlCDR3;-   CPA.7.011, CPA.7.011.VH, CPA.7.011.VL, CPA.7.011.HC, CPA.7.011.LC,    CPA.7.011.H1, CPA.7.011.H2, CPA.7.011.H3 CPA.7.011.H4;    CPA.7.011.vhCDR1, CPA.7.011.vhCDR2, CPA.7.011.vhCDR3,    CPA.7.011.vlCDR1, CPA.7.011.vlCDR2, and CPA.7.011.vlCDR3;-   CPA.7.012, CPA.7.012.VH, CPA.7.012.VL, CPA.7.012.HC, CPA.7.012.LC,    CPA.7.012.H1, CPA.7.012.H2, CPA.7.012.H3 CPA.7.012.H4;    CPA.7.012.vhCDR1, CPA.7.012.vhCDR2, CPA.7.012.vhCDR3,    CPA.7.012.vlCDR1, CPA.7.012.vlCDR2, and CPA.7.012.vlCDR3;-   CPA.7.013, CPA.7.013.VH, CPA.7.013.VL, CPA.7.013.HC, CPA.7.013.LC,    CPA.7.013.H1, CPA.7.013.H2, CPA.7.013.H3 CPA.7.013.H4;    CPA.7.013.vhCDR1, CPA.7.013.vhCDR2, CPA.7.013.vhCDR3,    CPA.7.013.vlCDR1, CPA.7.013.vlCDR2, and CPA.7.013.vlCDR3;-   CPA.7.014, CPA.7.014.VH, CPA.7.014.VL, CPA.7.014.HC, CPA.7.014.LC,    CPA.7.014.H1, CPA.7.014.H2, CPA.7.014.H3 CPA.7.014.H4;    CPA.7.014.vhCDR1, CPA.7.014.vhCDR2, CPA.7.014.vhCDR3,    CPA.7.014.vlCDR1, CPA.7.014.vlCDR2, and CPA.7.014.vlCDR3;-   CPA.7.015, CPA.7.015.VH, CPA.7.015.VL, CPA.7.015.HC, CPA.7.015.LC,    CPA.7.015.H1, CPA.7.015.H2, CPA.7.015.H3 CPA.7.015.H4;    CPA.7.015.vhCDR1, CPA.7.015.vhCDR2, CPA.7.015.vhCDR3,    CPA.7.015.vlCDR1, CPA.7.015.vlCDR2, and CPA.7.015.vlCDR3;-   CPA.7.017, CPA.7.017.VH, CPA.7.017.VL, CPA.7.017.HC, CPA.7.017.LC,    CPA.7.017H1, CPA.7.017.H2, CPA.7.017.H3 CPA.7.017.H4;    CPA.7.017.vhCDR1, CPA.7.000171.vhCDR2, CPA.7.017.vhCDR3,    CPA.7.017.vlCDR1, CPA.7.017.vlCDR2, and CPA.7.017.vlCDR3;-   CPA.7.018, CPA.7.018.VH, CPA.7.018.VL, CPA.7.018.HC, CPA.7.018.LC,    CPA.7.018.H1, CPA.7.018.H2, CPA.7.018.H3 CPA.7.018.H4;    CPA.7.017.vhCDR1, CPA.7.017.vhCDR2, CPA.7.017.vhCDR3,    CPA.7.017.vlCDR1, CPA.7.017.vlCDR2, and CPA.7.017.vlCDR3;-   CPA.7.019, CPA.7.019.VH, CPA.7.019.VL, CPA.7.019.HC, CPA.7.019.LC,    CPA.7.019.H1, CPA.7.019.H2, CPA.7.019.H3 CPA.7.019.H4;    CPA.7.019.vhCDR1, CPA.7.019.vhCDR2, CPA.7.019.vhCDR3,    CPA.7.019.vlCDR1, CPA.7.019.vlCDR2, and CPA.7.019.vlCDR3;-   CPA.7.021, CPA.7.021.VH, CPA.7.021.VL, CPA.7.021.HC, CPA.7.021.LC,    CPA.7.021.H1, CPA.7.021.H2, CPA.7.021.H3 CPA.7.021.H4;    CPA.7.021.vhCDR1, CPA.7.021.vhCDR2, CPA.7.021.vhCDR3,    CPA.7.021.vlCDR1, CPA.7.021.vlCDR2, and CPA.7.021.vlCDR3;-   CPA.7.022, CPA.7.022.VH, CPA.7.022.VL, CPA.7.022.HC, CPA.7.022.LC,    CPA.7.022.H1, CPA.7.022.H2, CPA.7.022.H3 CPA.7.022.H4;    CPA.7.022.vhCDR1, CPA.7.022.vhCDR2, CPA.7.002201.vhCDR3,    CPA.7.022.vlCDR1, CPA.7.022.vlCDR2, and CPA.7.022.vlCDR3;-   CPA.7.023, CPA.7.023.VH, CPA.7.023.VL, CPA.7.023.HC, CPA.7.023.LC,    CPA.7.023.H1, CPA.7.023.H2, CPA.7.023.H3 CPA.7.023.H4;    CPA.7.023.vhCDR1, CPA.7.023.vhCDR2, CPA.7.023.vhCDR3,    CPA.7.023.vlCDR1, CPA.7.023.vlCDR2, and CPA.7.023.vlCDR3;-   CPA.7.024, CPA.7.024.VH, CPA.7.024.VL, CPA.7.024.HC, CPA.7.024.LC,    CPA.7.024.H1, CPA.7.024.H2, CPA.7.024.H3 CPA.7.024.H4;    CPA.7.024.vhCDR1, CPA.7.024.vhCDR2, CPA.7.024.vhCDR3,    CPA.7.024.vlCDR1, CPA.7.024.vlCDR2, and CPA.7.024.vlCDR3;-   CPA.7.033, CPA.7.033.VH, CPA.7.033.VL, CPA.7.033.HC, CPA.7.033.LC,    CPA.7.033.H1, CPA.7.033.H2, CPA.7.033.H3 CPA.7.033.H4;    CPA.7.033.vhCDR1, CPA.7.033.vhCDR2, CPA.7.033.vhCDR3,    CPA.7.033.vlCDR1, CPA.7.033.vlCDR2, and CPA.7.033.vlCDR3;-   CPA.7.034, CPA.7.034.VH, CPA.7.034.VL, CPA.7.034.HC, CPA.7.034.LC,    CPA.7.034.H1, CPA.7.034.H2, CPA.7.034.H3 CPA.7.034.H4;    CPA.7.034.vhCDR1, CPA.7.034.vhCDR2, CPA.7.034.vhCDR3,    CPA.7.034.vlCDR1, CPA.7.034.vlCDR2, and CPA.7.034.vlCDR3;-   CPA.7.036, CPA.7.036.VH, CPA.7.036.VL, CPA.7.036.HC, CPA.7.036.LC,    CPA.7.036.H1, CPA.7.036.H2, CPA.7.036.H3 CPA.7.036.H4;    CPA.7.036.vhCDR1, CPA.7.036.vhCDR2, CPA.7.036.vhCDR3,    CPA.7.036.vlCDR1, CPA.7.036.vlCDR2, and CPA.7.036.vlCDR3;-   CPA.7.040, CPA.7.040.VH, CPA.7.040.VL, CPA.7.040.HC, CPA.7.040.LC,    CPA.7.040.H1, CPA.7.040.H2, CPA.7.040.H3 and CPA.7.040.H4;    CPA.7.040.vhCDR1, CPA.7.040.vhCDR2, CPA.7.040.vhCDR3,    CPA.7.040.vlCDR1, CPA.7.040.vlCDR2, and CPA.7.040.vlCDR3;-   CPA.7.046, CPA.7.046.VH, CPA.7.046.VL, CPA.7.046.HC, CPA.7.046.LC,    CPA.7.046.H1, CPA.7.046.H2, CPA.7.046.H3 CPA.7.046. H4;    CPA.7.046.vhCDR1, CPA.7.046.vhCDR2, CPA.7.046.vhCDR3,    CPA.7.046.vlCDR1, CPA.7.046.vlCDR2, and CPA.7.046.vlCDR3;-   CPA.7.047, CPA.7.047.VH, CPA.7.047.VL, CPA.7.047.HC, CPA.7.047.LC,    CPA.7.047.H1, CPA.7.047.H2, CPA.7.047.H3 CPA.7.047.H4;    CPA.7.047.vhCDR1, CPA.7.047.vhCDR2, CPA.7.047.vhCDR3,    CPA.7.047.vlCDR1, CPA.7.004701.vlCDR2, and CPA.7.047.vlCDR3;-   CPA.7.049, CPA.7.049.VH, CPA.7.049.VL, CPA.7.049.HC, CPA.7.049.LC,    CPA.7.049.H1, CPA.7.049.H2, CPA.7.049.H3 CPA.7.049.H4;    CPA.7.049.vhCDR1, CPA.7.049.vhCDR2, CPA.7.049.vhCDR3,    CPA.7.049.vlCDR1, CPA.7.049.vlCDR2, and CPA.7.049.vlCDR3; and-   CPA.7.050, CPA.7.050.VH, CPA.7.050.VL, CPA.7.050.HC, CPA.7.050.LC,    CPA.7.050.H1, CPA.7.050.H2, CPA.7.050.H3 CPA.7.050.H4,    CPA.7.050.vhCDR1, CPA.7.050.vhCDR2, CPA.7.050.vhCDR3,    CPA.7.050.vlCDR1, CPA.7.050.vlCDR2, and CPA.7.050.vlCDR3.-   CPA.7.028, CPA.7.028.VH, CPA.7.028.VL, CPA.7.028.HC, CPA.7.028.LC,    CPA.7.028.H1, CPA.7.028.H2, CPA.7.028.H3 and CPA.7.028.H4;    CPA.7.028.vhCDR1, CPA.7.028.vhCDR2, CPA.7.028.vhCDR3,    CPA.7.028.vlCDR1, CPA.7.028.vlCDR2, and CPA.7.028.vlCDR3.-   CPA.7.030, CPA.7.030.VH, CPA.7.030.VL, CPA.7.030.HC, CPA.7.030.LC,    CPA.7.030.H1, CPA.7.030.H2, CPA.7.030.H3 and CPA.7.030.H4;    CPA.7.030.vhCDR1, CPA.7.030.vhCDR2, CPA.7.030.vhCDR3,    CPA.7.030.vlCDR1, CPA.7.030.vlCDR2, and CPA.7.030.vlCDR3.-   CPA.7.041, CPA.7.041.VH, CPA.7.041.VL, CPA.7.041.HC, CPA.7.041.LC,    CPA.7.041.H1, CPA.7.041.H2, CPA.7.041.H3 and CPA.7.041.H4;    CPA.7.041.vhCDR1, CPA.7.041.vhCDR2, CPA.7.041.vhCDR3,    CPA.7.041.vlCDR1, CPA.7.041.vlCDR2, and CPA.7.041.vlCDR3.-   CPA.7.016, CPA.7.016.VH, CPA.7.016.VL, CPA.7.016.HC, CPA.7.016.LC,    CPA.7.016.H1, CPA.7.016.H2, CPA.7.016.H3 and CPA.7.016.H4;    CPA.7.016.vhCDR1, CPA.7.016.vhCDR2, CPA.7.016.vhCDR3,    CPA.7.016.vlCDR1, CPA.7.016.vlCDR2, and CPA.7.016.vlCDR3.-   CPA.7.020, CPA.7.020.VH, CPA.7.020.VL, CPA.7.020.HC, CPA.7.020.LC,    CPA.7.020.H1, CPA.7.020.H2, CPA.7.020.H3 and CPA.7.020.H4;    CPA.7.020.vhCDR1, CPA.7.020.vhCDR2, CPA.7.020.vhCDR3,    CPA.7.020.vlCDR1, CPA.7.020.vlCDR2, and CPA.7.020.vlCDR3.-   CPA.7.038, CPA.7.038.VH, CPA.7.038.VL, CPA.7.038.HC, CPA.7.038.LC,    CPA.7.038.H1, CPA.7.038.H2, CPA.7.038.H3 and CPA.7.038.H4;    CPA.7.038.vhCDR1, CPA.7.038.vhCDR2, CPA.7.038.vhCDR3,    CPA.7.038.vlCDR1, CPA.7.038.vlCDR2, and CPA.7.038.vlCDR3.-   CPA.7.044, CPA.7.044.VH, CPA.7.044.VL, CPA.7.044.HC, CPA.7.044.LC,    CPA.7.044.H1, CPA.7.044.H2, CPA.7.044.H3 and CPA.7.044.H4;    CPA.7.044.vhCDR1, CPA.7.044.vhCDR2, CPA.7.044.vhCDR3,    CPA.7.044.vlCDR1, CPA.7.044.vlCDR2, and CPA.7.044.vlCDR3.-   CPA.7.045, CPA.7.045.VH, CPA.7.045.VL, CPA.7.045.HC, CPA.7.045.LC,    CPA.7.045.H1, CPA.7.045.H2, CPA.7.045.H3 and CPA.7.045.H4;    CPA.7.045.vhCDR1, CPA.7.045.vhCDR2, CPA.7.045.vhCDR3,    CPA.7.045.vlCDR1, CPA.7.045.vlCDR2, and CPA.7.045.vlCDR3.

In some embodiments, the anti-TIGIT antibody is selected from anantibody the sequences for which are shown in FIG. 3 :

-   CPA.9.018, CPA.9.018.VH, CPA.9.018.VL, CPA.9.018.HC, CPA.9.018.LC,    CPA.9.018.H1, CPA.9.018.H2, CPA.9.018.H3, CPA.9.018.H4;    CPA.9.018.H4(S241P); CPA.9.018.vhCDR1, CPA.9.018.vhCDR2,    CPA.9.018.vhCDR3, CPA.9.018.vlCDR1, CPA.9.018.vlCDR2,    CPA.9.018.vlCDR3 and scFv-CPA.9.018;-   CPA.9.027, CPA.9.027.VH, CPA.9.027.VL, CPA.9.027.HC, CPA.9.027.LC,    CPA.9.027.H1, CPA.9.027.H2, CPA.9.027.H3, CPA.9.027.H4;    CPA.9.018.H4(S241P); CPA.9.027.vhCDR1, CPA.9.027.vhCDR2,    CPA.9.027.vhCDR3, CPA.9.027.vlCDR1, CPA.9.027.vlCDR2,    CPA.9.027.vlCDR3 and scFv-CPA.9.027;-   CPA.9.049, CPA.9.049.VH, CPA.9.049.VL, CPA.9.049.HC, CPA.9.049.LC,    CPA.9.049.H1, CPA.9.049.H2, CPA.9.049.H3; CPA.9.049.H4;    CPA.9.049.H4(S241P); CPA.9.049.vhCDR1, CPA.9.049.vhCDR2,    CPA.9.049.vhCDR3, CPA.9.049.vlCDR1, CPA.9.049.vlCDR2,    CPA.9.049.vlCDR3 and scFv-CPA.9.049;-   CPA.9.057, CPA.9.057.VH, CPA.9.057.VL, CPA.9.057.HC, CPA.9.057.LC,    CPA.9.057.H1, CPA.9.057.H2, CPA.9.057.H3; CPA.9.057.H4;    CPA.9.057.H4(S241P); CPA.9.057.vhCDR1, CPA.9.057.vhCDR2,    CPA.9.057.vhCDR3, CPA.9.057.vlCDR1, CPA.9.057.vlCDR2,    CPA.9.057.vlCDR3 and scFv-CPA.9.057;-   CPA.9.059, CPA.9.059.VH, CPA.9.059.VL, CPA.9.059.HC, CPA.9.059.LC,    CPA.9.059.H1, CPA.9.059.H2, CPA.9.059.H3; CPA.9.059.H4;    CPA.9.059.H4(S241P); CPA.9.059.vhCDR1, CPA.9.059.vhCDR2,    CPA.9.059.vhCDR3, CPA.9.059.vlCDR1, CPA.9.059.vlCDR2,    CPA.9.059.vlCDR3 and scFv-CPA.9.059;-   CPA.9.083, CPA.9.083.VH, CPA.9.083.VL, CPA.9.083.HC, CPA.9.083.LC,    CPA.9.083.H1, CPA.9.083.H2, CPA.9.083.H3; CPA.9.083.H4;    CPA.9.083.H4(S241P); CPA.9.083.vhCDR1, CPA.9.083.vhCDR2,    CPA.9.083.vhCDR3, CPA.9.083.vlCDR1, CPA.9.083.vlCDR2,    CPA.9.083.vlCDR3 and scFv-CPA.9.083;-   CPA.9.086, CPA.9.086.VH, CPA.9.086.VL, CPA.9.086.HC, CPA.9.086.LC,    CPA.9.086.H1, CPA.9.086.H2, CPA.9.086.H3; CPA.9.086.H4;    CPA.9.086.H4(S241P); CPA.9.086.vhCDR1, CPA.9.086.vhCDR2,    CPA.9.086.vhCDR3, CPA.9.086.vlCDR1, CPA.9.086.vlCDR2,    CPA.9.086.vlCDR3 and scFv-CPA.9.086;-   CPA.9.089, CPA.9.089.VH, CPA.9.089.VL, CPA.9.089.HC, CPA.9.089.LC,    CPA.9.089.H1, CPA.9.089.H2, CPA.9.089.H3; CPA.9.089.H4;    CPA.9.089.H4(S241P); CPA.9.089.vhCDR1, CPA.9.089.vhCDR2,    CPA.9.089.vhCDR3, CPA.9.089.vlCDR1, CPA.9.089.vlCDR2,    CPA.9.089.vlCDR3 and scFv-CPA.9.089;-   CPA.9.093, CPA.9.093.VH, CPA.9.093.VL, CPA.9.093.HC, CPA.9.093.LC,    CPA.9.093.H1, CPA.9.093.H2, CPA.9.093.H3; CPA.9.093.H4;    CPA.9.093.H4(S241P); CPA.9.093.vhCDR1, CPA.9.093.vhCDR2,    CPA.9.093.vhCDR3, CPA.9.093.vlCDR1, CPA.9.093.vlCDR2,    CPA.9.093.vlCDR3 and scFv-CPA.9.093;-   CPA.9.101, CPA.9.101.VH, CPA.9.101.VL, CPA.9.101.HC, CPA.9.101.LC,    CPA.9.101.H1, CPA.9.101.H2, CPA.9.101.H3; CPA.9.101.H4;    CPA.9.101.H4(S241P); CPA.9.101.vhCDR1, CPA.9.101.vhCDR2,    CPA.9.101.vhCDR3, CPA.9.101.vlCDR1, CPA.9.101.vlCDR2,    CPA.9.101.vlCDR3 and scFv-CPA.9.101; and-   CPA.9.103, CPA.9.103.VH, CPA.9.103.VL, CPA.9.103.HC, CPA.9.103.LC,    CPA.9.103.H1, CPA.9.103.H2, CPA.9.103.H3; CPA.9.103.H4;    CPA.9.103.H4(S241P); CPA.9.103.vhCDR1, CPA.9.103.vhCDR2,    CPA.9.103.vhCDR3, CPA.9.103.vlCDR1, CPA.9.103.vlCDR2,    CPA.9.103.vlCDR3 and scFv-CPA.9.103.-   CHA.9.536.1, CHA.9.536.1.VH, CHA.9.536.1.VL, CHA.9.536.1.HC,    CHA.9.536.1.LC, CHA.9.536.1.H1, CHA.9.536.1.H2, CHA.9.536.1.H3;    CHA.9.536.1.H4, CHA.9.536.1.H4(S241P), CHA.9.536.1.vhCDR1,    CHA.9.536.1.vhCDR2, CHA.9.536.1.vhCDR3, CHA.9.536.1.vlCDR1,    CHA.9.536.1.vlCDR2 and CHA.9.536.1.vhCDR3;-   CHA.9.536.3, CHA.9.536.3.VH, CHA.9.536.3.VL, CHA.9.536.3.HC,    CHA.9.536.3.LC, CHA.9.536.3.H1, CHA.9.536.3.H2, CHA.9.536.3.H3;    CHA.9.536.3.H4, CHA.9.536.3.H4(S241P); CHA.9.536.3.vhCDR1,    CHA.9.536.3.vhCDR2, CHA.9.536.3.vhCDR3, CHA.9.536.3.vlCDR1,    CHA.9.536.3.vlCDR2 and CHA.9.536.3.vhCDR3;-   CHA.9.536.4, CHA.9.536.4.VH, CHA.9.536.4.VL, CHA.9.536.4.HC,    CHA.9.536.4.LC, CHA.9.536.4.H1, CHA.9.536.4.H2, CHA.9.536.4.H3;    CHA.9.536.4.H4,-   CHA.9.536.4.H4(S241P), CHA.9.536.4.vhCDR1, CHA.9.536.4.vhCDR2,    CHA.9.536.4.vhCDR3, CHA.9.536.4.vlCDR1, CHA.9.536.4.vlCDR2 and    CHA.9.536.4.vhCDR3;-   CHA.9.536.5, CHA.9.536.5.VH, CHA.9.536.5.VL, CHA.9.536.5.HC,    CHA.9.536.5.LC, CHA.9.536.5.H1, CHA.9.536.5.H2, CHA.9.536.5.H3;    CHA.9.536.5.H4, CHA.9.536.5.H4(S241P), CHA.9.536.5.vhCDR1,    CHA.9.536.5.vhCDR2, CHA.9.536.5.vhCDR3, CHA.9.536.5.vlCDR1,    CHA.9.536.5.vlCDR2 and CHA.9.536.5.vhCDR3;-   CHA.9.536.6, CHA.9.536.6.VH, CHA.9.536.6.VL, CHA.9.536.6.HC,    CHA.9.536.6.LC, CHA.9.536.6.H1, CHA.9.536.6.H2, CHA.9.536.6.H3;    CHA.9.536.6.H4, CHA.9.536.6.vhCDR1, CHA.9.536.6.vhCDR2,    CHA.9.536.6.vhCDR3, CHA.9.536.6.vlCDR1, CHA.9.536.6.vlCDR2 and    CHA.9.536.6.vhCDR3;-   CHA.9.536.7, CHA.9.536.7.VH, CHA.9.536.7.VL, CHA.9.536.7.HC,    CHA.9.536.7.LC, CHA.9.536.7.H1, CHA.9.536.7.H2, CHA.9.536.7.H3;    CHA.9.536.7.H4, CHA.9.536.5.H4(S241P); CHA.9.536.7.vhCDR1,    CHA.9.536.7.vhCDR2, CHA.9.536.7.vhCDR3, CHA.9.536.7.vlCDR1,    CHA.9.536.7.vlCDR2 and CHA.9.536.7.vhCDR3;-   CHA.9.536.8, CHA.9.536.8.VH, CHA.9.536.8.VL, CHA.9.536.8.HC,    CHA.9.536.8.LC, CHA.9.536.8.H1, CHA.9.536.8.H2, CHA.9.536.8.H3;    CHA.9.536.8.H4, CHA.9.536.8.H4(S241P), CHA.9.536.8.vhCDR1,    CHA.9.536.8.vhCDR2, CHA.9.536.8.vhCDR3, CHA.9.536.8.vlCDR1,    CHA.9.536.8.vlCDR2 and CHA.9.536.8.vhCDR3;-   CHA.9.560.1, CHA. 9.560.1VH, CHA. 9.560.1.VL, CHA. 9.560.1.HC, CHA.    9.560.1.LC, CHA. 9.560.1.H1, CHA. 9.560.1.H2, CHA. 9.560.1.H3; CHA.    9.560.1.H4, CHA. 9.560.1.H4(S241P), CHA. 9.560.1.vhCDR1, CHA.    9.560.1.vhCDR2, CHA. 9.560.1.vhCDR3, CHA. 9.560.1.vlCDR1, CHA.    9.560.1.vlCDR2 and CHA. 9.560.1.vhCDR3;-   CHA.9.560.3, CHA. 9.560. 3VH, CHA. 9.560. 3.VL, CHA. 9.560. 3.HC,    CHA. 9.560. 3.LC, CHA. 9.560. 3.H1, CHA. 9.560. 3.H2, CHA. 9.560.    3.H3; CHA.9.560.3.H4, CHA.9.560.3.H4(S241P); CHA. 9.560. 3.vhCDR1,    CHA. 9.560. 3.vhCDR2, CHA. 9.560. 3.vhCDR3, CHA. 9.560. 3.vlCDR1,    CHA. 9.560. 3.vlCDR2 and CHA. 9.560. 3.vhCDR3;-   CHA.9.560.4, CHA. 9.560. 4VH, CHA. 9.560. 4.VL, CHA. 9.560. 4.HC,    CHA. 9.560. 4.LC, CHA. 9.560. 4.H1, CHA. 9.560. 4.H2, CHA. 9.560.    4.H3; CHA.9.560.4.H4, CHA.9.560.4.H4(S241P), CHA. 9.560. 4.vhCDR1,    CHA. 9.560. 4.vhCDR2, CHA. 9.560. 4.vhCDR3, CHA. 9.560. 4.vlCDR1,    CHA. 9.560. 4.vlCDR2 and CHA. 9.560. 4.vhCDR3;-   CHA.9.560.5, CHA. 9.560. 5VH, CHA. 9.560. 5.VL, CHA. 9.560. 5.HC,    CHA. 9.560. 5.LC, CHA. 9.560. 5.H1, CHA. 9.560. 5.H2, CHA. 9.560.    5.H3; CHA. 9.560. 5.H4, CHA. 9.560. 5.vhCDR1, CHA. 9.560. 5.vhCDR2,    CHA. 9.560. 5.vhCDR3, CHA. 9.560. 5.vlCDR1, CHA. 9.560. 5.vlCDR2 and    CHA. 9.560. 5.vhCDR3;-   CHA.9.560.6, CHA. 9.560. 6VH, CHA. 9.560. 6.VL, CHA. 9.560. 6.HC,    CHA. 9.560. 6.LC, CHA. 9.560. 6.H1, CHA. 9.560. 6.H2, CHA. 9.560.    6.H3; CHA.9.560.6.H4, CHA.9.560.6.H4(S241P), CHA. 9.560. 6.vhCDR1,    CHA. 9.560. 6.vhCDR2, CHA. 9.560. 6.vhCDR3, CHA. 9.560. 6.vlCDR1,    CHA. 9.560. 6.vlCDR2 and CHA. 9.560. 6.vhCDR3;-   CHA.9.560.7, CHA. 9.560. 7VH, CHA. 9.560. 7.VL, CHA. 9.560. 7.HC,    CHA. 9.560. 7.LC, CHA. 9.560. 7.H1, CHA. 9.560. 7.H2, CHA. 9.560.    7.H3; CHA.9.560.7.H4; CHA.9.560.7.H4(S241P); CHA. 9.560. 7.vhCDR1,    CHA. 9.560. 7.vhCDR2, CHA. 9.560. 7.vhCDR3, CHA. 9.560. 7.vlCDR1,    CHA. 9.560. 7.vlCDR2 and CHA. 9.560. 7.vhCDR3;-   CHA.9.560.8, CHA. 9.560. 8VH, CHA. 9.560. 8.VL, CHA. 9.560. 8.HC,    CHA. 9.560. 8.LC, CHA. 9.560. 8.H1, CHA. 9.560. 8.H2, CHA. 9.560.    8.H3; CHA.9.560.8.H4, CHA.9.560.8.H4(S241P); CHA. 9.560. 8.vhCDR1,    CHA. 9.560. 8.vhCDR2, CHA. 9.560. 8.vhCDR3, CHA. 9.560. 8.vlCDR1,    CHA. 9.560. 8.vlCDR2 and CHA. 9.560. 8.vhCDR3;-   CHA.9.546.1, CHA. 9. 546.1VH, CHA. 9. 546.1.VL, CHA. 9. 546.1.HC,    CHA. 9. 546.1.LC, CHA. 9. 546.1.H1, CHA. 9. 546.1.H2, CHA. 9.    546.1.H3; CHA.9.546.1.H4, CHA.9.546.1.H4(S241P), CHA. 9.    546.1.vhCDR1, CHA. 9. 546.1.vhCDR2, CHA. 9. 546.1.vhCDR3, CHA. 9.    546.1.vlCDR1, CHA. 9. 546.1.vlCDR2 and CHA. 9. 546.1.vhCDR3;-   CHA.9.547.1, CHA. 9. 547.1VH, CHA. 9. 547.1.VL, CHA. 9. 547.1.HC,    CHA. 9. 547.1.LC, CHA. 9. 547.1.H1, CHA. 9. 547.1.H2, CHA. 9.    547.1.H3; CHA.9.547.1.H4, CHA.9.547.1.H4(S241P), CHA. 9.    547.1.vhCDR1, CHA. 9. 547.1.vhCDR2, CHA. 9. 547.1.vhCDR3, CHA. 9.    547.1.vlCDR1, CHA. 9. 547.1.vlCDR2 and CHA. 9. 547.1.vhCDR3;-   CHA.9.547.2, CHA. 9. 547. 2VH, CHA. 9. 547. 2.VL, CHA. 9. 547. 2.HC,    CHA. 9. 547. 2.LC, CHA. 9. 547. 2.H1, CHA. 9. 547. 2.H2,    CHA. 9. 547. 2.H3; CHA.9.547.2.H4, CHA.9.547.2.H4(S241P),    CHA. 9. 547. 2.vhCDR1, CHA. 9. 547. 2.vhCDR2, CHA. 9. 547. 2.vhCDR3,    CHA. 9. 547. 2.vlCDR1, CHA. 9. 547. 2.vlCDR2 and CHA. 9. 547.    2.vhCDR3;-   CHA.9.547.3, CHA. 9. 547. 3VH, CHA. 9. 547. 3.VL, CHA. 9. 547. 3.HC,    CHA. 9. 547. 3.LC, CHA. 9. 547. 3.H1, CHA. 9. 547. 3.H2,    CHA. 9. 547. 3.H3; CHA.9.547.3.H4, CHA.9.547.3.H4(S241P),    CHA. 9. 547. 3.vhCDR1, CHA. 9.547. 3.vhCDR2, CHA. 9. 547. 3.vhCDR3,    CHA. 9. 547. 3.vlCDR1, CHA. 9. 547. 3.vlCDR2 and CHA. 9. 547.    3.vhCDR3;-   CHA.9.547.4, CHA. 9. 547. 4VH, CHA. 9. 547. 4.VL, CHA. 9. 547. 4.HC,    CHA. 9.547. 4.LC, CHA. 9. 547. 4.H1, CHA. 9. 547. 4.H2, CHA. 9. 547.    4.H3; CHA.9.547.4.H4, CHA.9.547.4.H4(S241P), CHA. 9. 547. 4.vhCDR1,    CHA. 9. 547. 4.vhCDR2, CHA. 9. 547. 4.vhCDR3, CHA. 9. 547. 4.vlCDR1,    CHA. 9. 547. 4.vlCDR2 and CHA. 9. 547. 4.vhCDR3;-   CHA.9.547.6, CHA. 9. 547.6 VH, CHA. 9. 547. 6.VL, CHA. 9. 547. 6.HC,    CHA. 9. 547. 6.LC, CHA. 9. 547. 6.H1, CHA. 9. 547. 6.H2,    CHA. 9. 547. 6.H3; CHA.9.547.6.H4, CHA.9.547.6.H4(S241P),    CHA. 9. 547. 6.vhCDR1, CHA. 9. 547. 6.vhCDR2, CHA. 9. 547. 6.vhCDR3,    CHA. 9. 547. 6.vlCDR1, CHA. 9. 547. 6.vlCDR2 and CHA. 9. 547.    6.vhCDR3;-   CHA.9.547.7, CHA. 9. 547. 7VH, CHA. 9. 547. 7.VL, CHA. 9. 547. 7.HC,    CHA. 9. 547. 7.LC, CHA. 9. 547. 7.H1, CHA. 9. 547. 7.H2,    CHA. 9. 547. 7.H3; CHA.9.547.7.H4, CHA.9.547.7.H4(S241P),    CHA. 9. 547. 7.vhCDR1, CHA. 9. 547. 7.vhCDR2, CHA. 9. 547. 7.vhCDR3,    CHA. 9. 547. 7.vlCDR1, CHA. 9. 547. 7.vlCDR2 and CHA. 9. 547.    7.vhCDR3;-   CHA.9.547.8, CHA. 9. 547. 8VH, CHA. 9. 547. 8.VL, CHA. 9. 547. 8.HC,    CHA.9.547.8.LC, CHA. 9. 547. 8.H1, CHA. 9. 547. 8.H2, CHA. 9. 547.    8.H3; CHA.9.547.8.H4, CHA.9.547.8.H4(S241P), CHA. 9. 547. 8.vhCDR1,    CHA. 9. 547. 8.vhCDR2, CHA. 9. 547. 8.vhCDR3, CHA. 9. 547. 8.vlCDR1,    CHA. 9. 547. 8.vlCDR2 and CHA. 9. 547. 8.vhCDR3;-   CHA.9.547.9, CHA.9.547.9, CHA.9.547.9VH, CHA.9.547.9.VL, CHA.9.    547.9.HC, CHA.9.547.9.LC, CHA.9.547.9.H1, CHA.9.547.9.H2,    CHA.9.547.9.H3; CHA.9.547.9.H4, CHA.9.547.9.H4,    CHA.9.547.9.H4(S241P), CHA.9.547.9.H4(S241P), CHA.9.547.9.vhCDR1,    CHA.9.547.9.vhCDR2, CHA.9.547.9.vhCDR3, CHA.9.547.9.vlCDR1,    CHA.9.547.9.vlCDR2 and CHA.9.547.9.vhCDR3;-   CHA.9.547.13, CHA.9.547.13, CHA.9.547. 13VH, CHA.9. 547.13.VL,    CHA.9. 547.13.HC, CHA. 9.547.13.LC, CHA. 9.547.13.H1,    CHA.9.547.13.H2, CHA.9. 547.13.H3; CHA.9.547.13.H4, CHA.9.547.13.H4,    CHA.9.547.13.H4(S241P), CHA.9.547.13.H4(S241P), CHA. 9.    547.13.vhCDR1, CHA.9.547.13.vhCDR2, CHA.9.547. 13.vhCDR3, CHA. 9.    547.13.vlCDR1, CHA. 9. 547.13.vlCDR2 and CHA. 9. 547. 13.vhCDR3;-   CHA.9.541.1, CHA. 9. 541.1.VH, CHA. 9. 541.1.VL, CHA. 9. 541.1.HC,    CHA. 9. 541.1.LC, CHA. 9. 541.1.H1, CHA. 9. 541.1.H2, CHA. 9.    541.1.H3; CHA.9.541.1.H4, CHA.9.541.1.H4(S241P), CHA. 9.    541.1.vhCDR1, CHA. 9. 541.1.vhCDR2, CHA. 9. 541.1.vhCDR3, CHA. 9.    541.1.vlCDR1, CHA. 9. 541.1.vlCDR2 and CHA. 9.541.1.vhCDR3;-   CHA.9.541.3, CHA. 9. 541. 3.VH, CHA. 9. 541. 3.VL, CHA. 9. 541.    3.HC, CHA. 9. 541. 3.LC, CHA. 9. 541. 3.H1, CHA. 9. 541. 3.H2,    CHA. 9. 541. 3.H3; CHA.9.541.3.H4, CHA.9.541.3.H4(S241P),    CHA. 9. 541. 3.vhCDR1, CHA. 9. 541. 3.vhCDR2, CHA. 9. 541. 3.vhCDR3,    CHA. 9. 541. 3.vlCDR1, CHA. 9. 541. 3.vlCDR2 and CHA. 9.541.    3.vhCDR3;-   CHA.9.541.4, CHA. 9. 541.4.VH, CHA. 9. 541. 4.VL, CHA. 9. 541. 4.HC,    CHA. 9. 541. 4.LC, CHA. 9. 541. 4.H1, CHA. 9. 541. 4.H2,    CHA. 9. 541. 4.H3; CHA.9.541.4.H4, CHA.9.541.4.H4(S241P),    CHA. 9. 541. 4.vhCDR1, CHA. 9. 541. 4.vhCDR2, CHA. 9. 541. 4.vhCDR3,    CHA. 9. 541. 4.vlCDR1, CHA. 9. 541. 4.vlCDR2 and CHA. 9.541.    4.vhCDR3;-   CHA.9.541.5, CHA. 9. 541. 5.VH, CHA. 9. 541. 5.VL, CHA. 9. 541.    5.HC, CHA. 9. 541. 5.LC, CHA. 9. 541. 5.H1, CHA. 9. 541. 5.H2,    CHA. 9. 541. 5.H3; CHA.9.541.5.H4, CHA.9.541.5.H4(S241P),    CHA. 9. 541. 5.vhCDR1, CHA. 9. 541. 5.vhCDR2, CHA. 9. 541. 5.vhCDR3,    CHA. 9. 541. 5.vlCDR1, CHA. 9. 541. 5.vlCDR2 and CHA. 9.541.    5.vhCDR3;-   CHA.9.541.6, CHA. 9. 541. 6.VH, CHA. 9. 541. 6.VL, CHA. 9. 541.    6.HC, CHA. 9. 541. 6.LC, CHA. 9. 541. 6.H1, CHA. 9. 541. 6.H2,    CHA. 9. 541.6.H3; CHA.9.541.6.H4, CHA.9.541.6.H4(S241P),    CHA. 9. 541. 6.vhCDR1, CHA. 9. 541. 6.vhCDR2, CHA. 9. 541. 6.vhCDR3,    CHA. 9. 541. 6.vlCDR1, CHA. 9. 541. 6.vlCDR2 and CHA. 9.541.    6.vhCDR3;-   CHA.9.541.7, CHA. 9. 541. 7.VH, CHA. 9. 541. 7.VL, CHA. 9. 541.    7.HC, CHA. 9. 541. 7.LC, CHA. 9. 541. 7.H1, CHA. 9. 541. 7.H2,    CHA. 9. 541. 7.H3; CHA.9.541.7.H4, CHA.9.541.7.H4(S241P),    CHA. 9. 541. 7.vhCDR1, CHA. 9. 541. 7.vhCDR2, CHA. 9. 541. 7.vhCDR3,    CHA. 9. 541. 7.vlCDR1, CHA. 9. 541. 7.vlCDR2 and CHA. 9.541.    7.vhCDR3; and-   CHA.9.541.8, CHA. 9. 541. 8.VH, CHA. 9. 541. 8.VL, CHA. 9. 541.    8.HC, CHA. 9. 541. 8.LC, CHA. 9. 541. 8.H1, CHA. 9. 541. 8.H2,    CHA. 9. 541. 8.H3; CHA.9.541.8.H4, CHA.9.541.8.H4(S241P);    CHA. 9. 541. 8vhCDR1, CHA. 9. 541. 8.vhCDR2, CHA. 9. 541. 8.vhCDR3,    CHA. 9. 541. 8.vlCDR1, CHA. 9. 541. 8.vlCDR2 and CHA. 9.541.    8.vhCDR3.

In some embodiments, the anti-TIGIT antibody is CPA.9.083.H4(S241P), theanti-PD-1 antibody is pembrolizumab, and the anti-PVRIG antibody is oneof the above and/or from FIG. 5 or 63 .

In some embodiments, the anti-TIGIT antibody is CPA.9.086.H4(S241P), theanti-PD-1 antibody is pembrolizumab, and the anti-PVRIG antibody is oneof the above and/or from FIG. 5 or 63 .

In some embodiments, the anti-TIGIT antibody is CHA.9.547.7.H4(S241P),the anti-PD-1 antibody is pembrolizumab, and the anti-PVRIG antibody isone of the above and/or from FIG. 5 or 63 .

In some embodiments, the anti-TIGIT antibody is CHA.9.547.13.H4(S241P),the anti-PD-1 antibody is pembrolizumab, and the anti-PVRIG antibody isone of the above and/or from FIG. 5 or 63 .

In some embodiments, the anti-TIGIT antibody is CPA.9.083.H4(S241P), theanti-PD-1 antibody is nivolumab, and the anti-PVRIG antibody is one ofthe above and/or from FIG. 5 or 63 .

In some embodiments, the anti-TIGIT antibody is CPA.9.086.H4(S241P), theanti-PD-1 antibody is nivolumab, and the anti-PVRIG antibody is one ofthe above and/or from FIG. 5 or 63 .

In some embodiments, the anti-TIGIT antibody is CHA.9.547.7.H4(S241P),the anti-PD-1 antibody is nivolumab, and the anti-PVRIG antibody is oneof the above and/or from FIG. 5 or 63 .

In some embodiments, the anti-TIGIT antibody is CHA.9.547.13.H4(S241P),the PD-1 antibody is nivolumab, and the anti-PVRIG antibody is one ofthe above and/or from FIG. 5 or 63 .

In some embodiments, the anti-TIGIT antibody is CPA.9.083.H4(S241P), theanti-PD-1 antibody is cemiplimab, and the anti-PVRIG antibody is one ofthe above and/or from FIG. 5 or 63 .

In some embodiments, the anti-TIGIT antibody is CPA.9.086.H4(S241P), theanti-PD-1 antibody is cemiplimab, and the anti-PVRIG antibody is one ofthe above and/or from FIG. 5 or 63 .

In some embodiments, the anti-TIGIT antibody is CHA.9.547.7.H4(S241P),the anti-PD-1 antibody is cemiplimab, and the anti-PVRIG antibody is oneof the above and/or from FIG. 5 or 63 .

In some embodiments, the anti-TIGIT antibody is CHA.9.547.13.H4(S241P),the PD-1 antibody is cemiplimab, and the anti-PVRIG antibody is one ofthe above and/or from FIG. 5 or 63 .

In some embodiments, the anti-PVRIG antibody is CHA.7.518.1.H4(S241P),the anti-PD-1 antibody is pembrolizumab, and the anti-TIGIT antibody oneof the above and/or from FIG. 3 .

In some embodiments, the anti-PVRIG antibody is CHA.7.538.1.2.H4(S241P),the anti-PD-1 antibody is pembrolizumab, and the anti-TIGIT antibody oneof the above and/or from FIG. 3 .

In some embodiments, the anti-PVRIG antibody is CHA.7.518.1.H4(S241P),the anti-PD-1 antibody is nivolumab, and the anti-TIGIT antibody one ofthe above and/or from FIG. 3 .

In some embodiments, the anti-PVRIG antibody is CHA.7.538.1.2.H4(S241P),the anti-PD-1 antibody is nivolumab, and the anti-TIGIT antibody one ofthe above and/or from FIG. 3 .

In some embodiments, the anti-PVRIG antibody is CHA.7.518.1.H4(S241P),the anti-PD-1 antibody is cemiplimab, and the anti-TIGIT antibody one ofthe above and/or from FIG. 3 .

In some embodiments, the anti-PVRIG antibody is CHA.7.538.1.2.H4(S241P),the anti-PD-1 antibody is cemiplimab, and the anti-TIGIT antibody one ofthe above and/or from FIG. 3 .

In some embodiments, the anti-TIGIT antibody is CPA.9.083.H4(S241P), theanti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-1antibody is pembrolizumab.

In some embodiments, the anti-TIGIT antibody is CPA.9.083.H4(S241P), theanti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-1 antibodyis pembrolizumab.

In some embodiments, the anti-TIGIT antibody is CPA.9.086.H4(S241P), theanti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-1antibody is pembrolizumab.

In some embodiments, the anti-TIGIT antibody is CPA.9.086.H4(S241P), theanti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-1 antibodyis pembrolizumab.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.7.H4(S241P),the anti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-1antibody is pembrolizumab.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.7.H4(S241P),the anti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-1antibody is pembrolizumab.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.13.H4(S241P),the anti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-1antibody is pembrolizumab.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.13.H4(S241P),the anti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-1antibody is pembrolizumab.

In some embodiments, the anti-TIGIT antibody is CPA.9.083.H4(S241P), theanti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-1antibody is SHR-1210.

In some embodiments, the anti-TIGIT antibody is CPA.9.083.H4(S241P), theanti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-1 antibodyis SHR-1210.

In some embodiments, the anti-TIGIT antibody is CPA.9.086.H4(S241P), theanti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-1antibody is SHR-1210.

In some embodiments, the anti-TIGIT antibody is CPA.9.086.H4(S241P), theanti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-1 antibodyis SHR-1210.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.7.H4(S241P),the anti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-1antibody is SHR-1210.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.7.H4(S241P),the anti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-1antibody is SHR-1210.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.13.H4(S241P),the anti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-1antibody is SHR-1210.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.13.H4(S241P),the anti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-1antibody is SHR-1210.

In some embodiments, the anti-TIGIT antibody is CPA.9.083.H4(S241P), theanti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-1antibody is IBI308.

In some embodiments, the anti-TIGIT antibody is CPA.9.083.H4(S241P), theanti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-1 antibodyis IBI308.

In some embodiments, the anti-TIGIT antibody is CPA.9.086.H4(S241P), theanti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-1antibody is IBI308.

In some embodiments, the anti-TIGIT antibody is CPA.9.086.H4(S241P), theanti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-1 antibodyis IBI308.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.7.H4(S241P),the anti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-1antibody is IBI308.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.7.H4(S241P),the anti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-1antibody is IBI308.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.13.H4(S241P),the anti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-1antibody is IBI308.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.13.H4(S241P),the anti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-1antibody is IBI308.

In some embodiments, the anti-TIGIT antibody is CPA.9.083.H4(S241P), theanti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-1antibody is BGB-A317.

In some embodiments, the anti-TIGIT antibody is CPA.9.083.H4(S241P), theanti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-1 antibodyis BGB-A317.

In some embodiments, the anti-TIGIT antibody is CPA.9.086.H4(S241P), theanti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-1antibody is BGB-A317.

In some embodiments, the anti-TIGIT antibody is CPA.9.086.H4(S241P), theanti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-1 antibodyis BGB-A317.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.7.H4(S241P),the anti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-1antibody is BGB-A317.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.7.H4(S241P),the anti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-1antibody is BGB-A317.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.13.H4(S241P),the anti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-1antibody is BGB-A317.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.13.H4(S241P),the anti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-1antibody is BGB-A317.

In some embodiments, the anti-TIGIT antibody is CPA.9.083.H4(S241P), theanti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-1antibody is an anti-PD-1 antibody as recited in U.S. Patent PublicationNo. 2017/0081409.

In some embodiments, the anti-TIGIT antibody is CPA.9.083.H4(S241P), theanti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-1 antibodyis an anti-PD-1 antibody as recited in U.S. Patent Publication No.2017/0081409.

In some embodiments, the anti-TIGIT antibody is CPA.9.086.H4(S241P), theanti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-1antibody is an anti-PD-1 antibody as recited in U.S. Patent PublicationNo. 2017/0081409.

In some embodiments, the anti-TIGIT antibody is CPA.9.086.H4(S241P), theanti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-1 antibodyis an anti-PD-1 antibody as recited in U.S. Patent Publication No.2017/0081409.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.7.H4(S241P),the anti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-1antibody is an anti-PD-1 antibody as recited in U.S. Patent PublicationNo. 2017/0081409.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.7.H4(S241P),the anti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-1antibody is an anti-PD-1 antibody as recited in U.S. Patent PublicationNo. 2017/0081409.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.13.H4(S241P),the anti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-1antibody is an anti-PD-1 antibody as recited in U.S. Patent PublicationNo. 2017/0081409.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.13.H4(S241P),the anti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-1antibody is an anti-PD-1 antibody as recited in U.S. Patent PublicationNo. 2017/0081409.

In some embodiments, the anti-TIGIT antibody is CPA.9.083.H4(S241P), theanti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-1antibody is cemiplimab.

In some embodiments, the anti-TIGIT antibody is CPA.9.083.H4(S241P), theanti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-1 antibodyis cemiplimab.

In some embodiments, the anti-TIGIT antibody is CPA.9.086.H4(S241P), theanti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-1antibody is cemiplimab.

In some embodiments, the anti-TIGIT antibody is CPA.9.086.H4(S241P), theanti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-1 antibodyis cemiplimab.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.7.H4(S241P),the anti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-1antibody is cemiplimab.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.7.H4(S241P),the anti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-1antibody is cemiplimab.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.13.H4(S241P),the anti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-1antibody is cemiplimab.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.13.H4(S241P),the anti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-1antibody is cemiplimab.

In some embodiments, the anti-TIGIT antibody is CPA.9.083.H4(S241P), theanti-PD-L1 antibody is atezolizumab, and the anti-PVRIG antibody is oneof the above and/or from FIG. 5 or 63 .

In some embodiments, the anti-TIGIT antibody is CPA.9.086.H4(S241P theanti-PD-L1 antibody is atezolizumab, and the anti-PVRIG antibody is oneof the above and/or from FIG. 5 or 63 .

In some embodiments, the anti-TIGIT antibody is CHA.9.547.7.H4(S241P),the anti-PD-L1 antibody is atezolizumab, and the anti-PVRIG antibody isone of the above and/or from FIG. 5 or 63 .

In some embodiments, the anti-TIGIT antibody is CHA.9.547.13.H4(S241P),the anti-PD-L1 antibody is atezolizumab, and the anti-PVRIG antibody isone of the above and/or from FIG. 5 or 63 .

In some embodiments, the anti-TIGIT antibody is CPA.9.083.H4(S241P), theanti-PD-L1 antibody is avelumab, and the anti-PVRIG antibody is one ofthe above and/or from FIG. 5 or 63 .

In some embodiments, the anti-TIGIT antibody is CPA.9.086.H4(S241P), theanti-PD-L1 antibody is avelumab, and the anti-PVRIG antibody is one ofthe above and/or from FIG. 5 or 63 .

In some embodiments, the anti-TIGIT antibody is CHA.9.547.7.H4(S241P),the anti-PD-L1 antibody is avelumab, and the anti-PVRIG antibody is oneof the above and/or from FIG. 5 or 63 .

In some embodiments, the anti-TIGIT antibody is CHA.9.547.13.H4(S241Pthe anti-PD-L1 antibody is avelumab, and the anti-PVRIG antibody is oneof the above and/or from FIG. 5 or 63 .

In some embodiments, the anti-TIGIT antibody is CPA.9.083.H4(S241P), theanti-PD-L1 antibody is durvalumab, and the anti-PVRIG antibody is one ofthe above and/or from FIG. 5 or 63 .

In some embodiments, the anti-TIGIT antibody is CPA.9.086.H4(S241P theanti-PD-L1 antibody is durvalumab, and the anti-PVRIG antibody is one ofthe above and/or from FIG. 5 or 63 .

In some embodiments, the anti-TIGIT antibody is CHA.9.547.7.H4(S241P),the anti-PD-L1 antibody is durvalumab, and the anti-PVRIG antibody isone of the above and/or from FIG. 5 or 63 .

In some embodiments, the anti-TIGIT antibody is CHA.9.547.13.H4(S241P),the anti-PD-L1 antibody is durvalumab, and the anti-PVRIG antibody isone of the above and/or from FIG. 5 or 63 .

In some embodiments, the anti-PVRIG antibody is CHA.7.518.1.H4(S241P),the anti-PD-L1 antibody is atezolizumab, and the anti-TIGIT antibody oneof the above and/or from FIG. 3 .

In some embodiments, the anti-PVRIG antibody is CHA.7.538.1.2.H4(S241P),the anti-PD-L1 antibody is atezolizumab, and the anti-TIGIT antibody oneof the above and/or from FIG. 3 .

In some embodiments, the anti-PVRIG antibody is CHA.7.518.1.H4(S241P),the anti-PD-L1 antibody is atezolizumab, and the anti-TIGIT antibody oneof the above and/or from FIG. 3 .

In some embodiments, the anti-PVRIG antibody is CHA.7.538.1.2.H4(S241P),the anti-PD-L1 antibody is atezolizumab, and the anti-TIGIT antibody oneof the above and/or from FIG. 3 .

In some embodiments, the anti-PVRIG antibody is CHA.7.518.1.H4(S241P),the anti-PD-L1 antibody is avelumab, and the anti-TIGIT antibody one ofthe above and/or from FIG. 3 .

In some embodiments, the anti-PVRIG antibody is CHA.7.538.1.2.H4(S241P),the anti-PD-L1 antibody is avelumab, and the anti-TIGIT antibody one ofthe above and/or from FIG. 3 .

In some embodiments, the anti-PVRIG antibody is CHA.7.518.1.H4(S241P),the anti-PD-L1 antibody is avelumab, and the anti-TIGIT antibody one ofthe above and/or from FIG. 3 .

In some embodiments, the anti-PVRIG antibody is CHA.7.538.1.2.H4(S241P),the anti-PD-L1 antibody is avelumab, and the anti-TIGIT antibody one ofthe above and/or from FIG. 3 .

In some embodiments, the anti-PVRIG antibody is CHA.7.518.1.H4(S241P),the anti-PD-L1 antibody is durvalumab, and the anti-TIGIT antibody oneof the above and/or from FIG. 3 .

In some embodiments, the anti-PVRIG antibody is CHA.7.538.1.2.H4(S241P),the anti-PD-L1 antibody is durvalumab, and the anti-TIGIT antibody oneof the above and/or from FIG. 3 .

In some embodiments, the anti-PVRIG antibody is CHA.7.518.1.H4(S241P),the anti-PD-L1 antibody is durvalumab, and the anti-TIGIT antibody oneof the above and/or from FIG. 3 .

In some embodiments, the anti-PVRIG antibody is CHA.7.538.1.2.H4(S241P),the anti-PD-L1 antibody is durvalumab, and the anti-TIGIT antibody oneof the above and/or from FIG. 3 .

In some embodiments, the anti-TIGIT antibody is CPA.9.083.H4(S241P), theanti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-L1antibody is atezolizumab.

In some embodiments, the anti-TIGIT antibody is CPA.9.083.H4(S241P), theanti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-L1antibody is atezolizumab.

In some embodiments, the anti-TIGIT antibody is CPA.9.086.H4(S241P), theanti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-L1antibody is atezolizumab.

In some embodiments, the anti-TIGIT antibody is CPA.9.086.H4(S241P), theanti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-L1antibody is atezolizumab.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.7.H4(S241P),the anti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-L1antibody is atezolizumab.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.7.H4(S241P),the anti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-L1antibody is atezolizumab.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.13.H4(S241P),the anti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-L1antibody is atezolizumab.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.13.H4(S241P),the anti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-L1antibody is atezolizumab.

In some embodiments, the anti-TIGIT antibody is CPA.9.083.H4(S241P), theanti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-L1antibody is avelumab.

In some embodiments, the anti-TIGIT antibody is CPA.9.083.H4(S241P), theanti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-L1antibody is avelumab.

In some embodiments, the anti-TIGIT antibody is CPA.9.086.H4(S241P), theanti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-L1antibody is avelumab.

In some embodiments, the anti-TIGIT antibody is CPA.9.086.H4(S241P), theanti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-L1antibody is avelumab.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.7.H4(S241P),the anti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-L1antibody is avelumab.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.7.H4(S241P),the anti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-L1antibody is avelumab.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.13.H4(S241P),the anti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-L1antibody is avelumab.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.13.H4(S241P),the anti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-L1antibody is avelumab.

In some embodiments, the anti-TIGIT antibody is CPA.9.083.H4(S241P), theanti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-L1antibody is durvalumab.

In some embodiments, the anti-TIGIT antibody is CPA.9.083.H4(S241P), theanti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-L1antibody is durvalumab.

In some embodiments, the anti-TIGIT antibody is CPA.9.086.H4(S241P), theanti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-L1antibody is durvalumab.

In some embodiments, the anti-TIGIT antibody is CPA.9.086.H4(S241P), theanti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-L1antibody is durvalumab.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.7.H4(S241P),the anti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-L1antibody is durvalumab.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.7.H4(S241P),the anti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-L1antibody is durvalumab.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.13.H4(S241P),the anti-PVRIG antibody is CHA.7.538.1.2.H4(S241P), and the anti-PD-L1antibody is durvalumab.

In some embodiments, the anti-TIGIT antibody is CHA.9.547.13.H4(S241P),the anti-PVRIG antibody is CHA.7.518.1.H4(S241P), and the anti-PD-L1antibody is durvalumab.

B. Biomarker Analysis

As shown herein, the selection of the combination therapy to administercan be done using an evaluation of the expression of particularbiomarkers from tumor biopsy. That is, by taking a biopsy from a tumorsample of a patient and testing for the presence and levels of certainproteins using protein staining and sorting, a suitable therapy can bechosen. As shown in Example 2, cells from tumors can be screened toidentify immune and non-immune cell populations, and then the immunecell populations assessed for the levels of a number of biomarkersincluding PD-1, PD-L1, PVRIG, PVR, PVRL2 and TIGIT, including byexamining both ligand and antigen levels.

Thus, for example, to identify immune cell populations, antibodies toone or more of CD45, CD3, CD8, CD33, CD25, CD127, CD14, CD4 and CD56 canbe assessed to categorize the cell populations in the tumor sample asshown below in Table 1:

Cell Subset Name Gating Markers CD4⁺ T cells CD45⁺CD3⁺CD14⁻CD4⁺ CD8⁺ Tcells CD45⁺CD3⁺CD14⁻CD8⁺ CD4⁻CD8⁻ T cells CD45⁺CD3⁺CD14⁻CD4⁻CD8⁻ NKcells CD45⁺CD3⁻CD14⁻CD56⁺ Monocytes CD45⁺CD3⁻CD14⁺ mDCsCD45⁺CD3⁻CD14⁻CD56⁻CD33^(hi) pDCs CD45⁺CD3⁻CD14⁻CD56⁻CD33^(mid) CD45⁻cells CD45⁻

Several of these cell types are then assessed for expression of one ormore of PD-1, PD-L1, PVRIG, PVR, PVRL2 and TIGIT, generally usinglabeled antibodies and scored. If the percentage of PD-L1 positive tumorcells or immune cells is greater than 1% (>1%) compared to the sametumor cells stained with antibody relevant isotype control antibody forthe antibodies used, then a triple combination of anti-TIGIT, anti-PVRIGand anti-PD1 antibodies should be administered. Whereas, patients with afrequency of PD-L1 positive tumor cells or immune cells below 1% (<1%)compared to the isotype control should be administered a doublecombination of anti-TIGIT and anti-PVRIG antibodies.

1. Combination Therapy of Anti-TIGIT, Anti-PVRIG and Anti-PD-1

Antibodies

In some embodiments, once the immune cells from the tumor have beenoptionally tested for expression of at least one cell surface markerselected from PD-1, PD-L1, PVRIG, PVR, PVRL2 and TIGIT, therapeuticdecisions can be made. In the case where the expression of PD-L1positive tumor cells or immune cells is >1%, the patient can beadministered a triple combination of anti-TIGIT, anti-PVRIG andanti-PD-1 antibodies as outlined herein.

Accordingly, in one embodiment, antibodies containing the CDR sets fromthe anti-TIGIT antibody CPA.9.083 are combined with antibodiescontaining the CDR sets from the anti-PVRIG antibody CHA.7.518.1 andpembrolizumab. In a particular embodiment, CPA.9.083.H4(S241P) iscombined with CHA.7.518.1.H4(S241P) and pembrolizumab.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CPA.9.083 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.538.1.2 andpembrolizumab. In a particular embodiment, CPA.9.083.H4(S241P) iscombined with CHA.7.538.1.2.H4(S241P) and pembrolizumab.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CPA.9.086 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.518.1 and pembrolizumab.In a particular embodiment, CPA.9.086.H4(S241P is combined withCHA.7.518.1.H4(S241P) and pembrolizumab.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CPA.9.086 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.538.1.2 andpembrolizumab. In a particular embodiment, CPA.9.086.H4(S241P) iscombined with CHA.7.538.1.2.H4(S241P) and pembrolizumab.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CHA.9.547.7 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.518.1 and pembrolizumab.In a particular embodiment, CHA.9.547.7H4(S241P) is combined withCHA.7.518.1.H4(S241P) and pembrolizumab.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CHA.9.547.7 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.538.1.2 andpembrolizumab. In a particular embodiment, CHA.9.547.7.H4(S241P) iscombined with CHA.7.538.1.2.H4(S241P) and pembrolizumab.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CHA.9.547.13 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.518.1 and pembrolizumab.In a particular embodiment, CHA.9.547.13.H4(S241P) is combined withCHA.7.518.1.H4(S241P) and pembrolizumab.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CHA.9.547.13 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.538.1.2 andpembrolizumab. In a particular embodiment, CHA.9.547.13.H4(S241P) iscombined with CHA.7.538.1.2.H4(S241P) and pembrolizumab.

Accordingly, in one embodiment, antibodies containing the CDR sets fromthe anti-TIGIT antibody CPA.9.083 are combined with antibodiescontaining the CDR sets from the anti-PVRIG antibody CHA.7.518.1 andnivolumab. In a particular embodiment, CPA.9.083.H4(S241P) is combinedwith CHA.7.518.1.H4(S241P) and nivolumab.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CPA.9.083 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.538.1.2 and nivolumab.In a particular embodiment, CPA.9.083.H4(S241P) is combined withCHA.7.538.1.2.H4(S241P) and nivolumab.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CPA.9.086 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.518.1 and nivolumab. Ina particular embodiment, CPA.9.086.H4(S241P is combined withCHA.7.518.1.H4(S241P) and nivolumab.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CPA.9.086 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.538.1.2 and nivolumab.In a particular embodiment, CPA.9.086.H4(S241P) is combined withCHA.7.538.1.2.H4(S241P) and nivolumab.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CHA.9.547.7 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.518.1 and nivolumab. Ina particular embodiment, CHA.9.547.7H4(S241P) is combined withCHA.7.518.1.H4(S241P) and nivolumab.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CHA.9.547.7 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.538.1.2 and nivolumab.In a particular embodiment, CHA.9.547.7.H4(S241P) is combined withCHA.7.538.1.2.H4(S241P) and nivolumab.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CHA.9.547.13 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.518.1 and nivolumab. Ina particular embodiment, CHA.9.547.13.H4(S241P) is combined withCHA.7.518.1.H4(S241P) and nivolumab.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CHA.9.547.13 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.538.1.2 and nivolumab.In a particular embodiment, CHA.9.547.13.H4(S241P) is combined withCHA.7.538.1.2.H4(S241P) and nivolumab.

Accordingly, in one embodiment, antibodies containing the CDR sets fromthe anti-TIGIT antibody CPA.9.083 are combined with antibodiescontaining the CDR sets from the anti-PVRIG antibody CHA.7.518.1 andcemiplimab. In a particular embodiment, CPA.9.083.H4(S241P) is combinedwith CHA.7.518.1.H4(S241P) and cemiplimab.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CPA.9.083 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.538.1.2 and cemiplimab.In a particular embodiment, CPA.9.083.H4(S241P) is combined withCHA.7.538.1.2.H4(S241P) and cemiplimab.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CPA.9.086 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.518.1 and cemiplimab. Ina particular embodiment, CPA.9.086.H4(S241P is combined withCHA.7.518.1.H4(S241P) and cemiplimab.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CPA.9.086 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.538.1.2 and cemiplimab.In a particular embodiment, CPA.9.086.H4(S241P) is combined withCHA.7.538.1.2.H4(S241P) and cemiplimab.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CHA.9.547.7 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.518.1 and cemiplimab. Ina particular embodiment, CHA.9.547.7H4(S241P) is combined withCHA.7.518.1.H4(S241P) and cemiplimab.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CHA.9.547.7 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.538.1.2 and cemiplimab.In a particular embodiment, CHA.9.547.7.H4(S241P) is combined withCHA.7.538.1.2.H4(S241P) and cemiplimab.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CHA.9.547.13 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.518.1 and cemiplimab. Ina particular embodiment, CHA.9.547.13.H4(S241P) is combined withCHA.7.518.1.H4(S241P) and cemiplimab.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CHA.9.547.13 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.538.1.2 and cemiplimab.In a particular embodiment, CHA.9.547.13.H4(S241P) is combined withCHA.7.538.1.2.H4(S241P) and cemiplimab.

2. Combination Therapy of Anti-TIGIT and Anti-PVRIG Antibodies

Similarly, once the once the immune cells from the tumor have beentested for expression of at least one cell surface marker selected fromPD-1, PD-L1, PVRIG, PVR, PVRL2 and TIGIT, therapeutic decisions can bemade. In the case where the expression of PD-L1 positive tumor cells orimmune cells is <1%, the patient can be administered a doublecombination of anti-TIGIT and anti-PVRIG antibodies as outlined herein.

Accordingly, in one embodiment, antibodies containing the CDR sets fromthe anti-TIGIT antibody CPA.9.083 are combined with antibodiescontaining the CDR sets from the anti-PVRIG antibody CHA.7.518.1. In aparticular embodiment, CPA.9.083.H4(S241P) is combined withCHA.7.518.1.H4(S241P).

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CPA.9.083 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.538.1.2. In a particularembodiment, CPA.9.083.H4(S241P) is combined withCHA.7.538.1.2.H4(S241P).

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CPA.9.086 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.518.1. In a particularembodiment, CPA.9.086.H4(S241P is combined with CHA.7.518.1.H4(S241P).

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CPA.9.086 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.538.1.2. In a particularembodiment, CPA.9.086.H4(S241P) is combined withCHA.7.538.1.2.H4(S241P).

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CHA.9.547.7 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.518.1. In a particularembodiment, CHA.9.547.7H4(S241P) is combined with CHA.7.518.1.H4(S241P).

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CHA.9.547.7 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.538.1.2. In a particularembodiment, CHA.9.547.7.H4(S241P) is combined withCHA.7.538.1.2.H4(S241P).

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CHA.9.547.13 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.518.1. In a particularembodiment, CHA.9.547.13.H4(S241P) is combined withCHA.7.518.1.H4(S241P).

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CHA.9.547.13 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.538.1.2. In a particularembodiment, CHA.9.547.13.H4(S241P) is combined withCHA.7.538.1.2.H4(S241P).

In one embodiment, the invention provides combinations of the anti-TIGITantibodies of the invention and anti-PD-1 antibodies. In one embodiment,the invention provides combinations of the anti-TIGIT antibodies of theinvention and anti-PD-L1 antibodies.

In one embodiment, a biopsy is taken from a tumor from a patient withcancer, and dissociated as is known in the art for FACS analysis. Thecells are stained with labeled antibodies to (1) TIGIT (for exampleusing any described herein or others in the art such as MBSA43); (2)PD-1 (for example using those known in the art including EH12.2H7,Keytruda®, Opdivo®, Cemiplimab, etc.); (3) PD-L1 (for example usingthose known in the art such as BM-1, atezolizumab, avelumab, anddurvalumab, outlined herein) and (4) PVR (for example using those knownin the art such as SKII.4); and (5) a relevant isotype control antibodyfor the antibodies used. FACS is done, and for each receptor, thepercentage of the cells expressing the receptor relative to the controlantibody is calculated. If the percentage of positive cells for TIGIT,PD-1, PD-1 and PVR is >1% for all 4 receptors, then the patient istreated with antibodies to TIGIT and PD-1 as outlined herein.

In one embodiment, a biopsy is taken from a tumor from a patient withcancer, and dissociated as is known in the art for FACS analysis. Thecells are stained with labeled antibodies to (1) PVRIG (generally usingCHA.7.518.1H4(S241P), for example, although any outlined inWO2016/134333 (specifically including any that bind, even if they don'tblock) or WO2017/041004) can be used); (2) PD-1 (for example using thoseknown in the art including EH12.2H7, Keytruda®, Opdivo®, Cemiplimab,etc.); (3) PD-L1 (for example using those known in the art such as BM-1,atezolizumab, avelumab, and durvalumab, outlined herein) and (4) PVRL2(for example using those known in the art such as TX11); and (5) arelevant isotype control antibody for the antibodies used. FACS is done,and for each receptor, the percentage of the cells expressing thereceptor relative to the control antibody is calculated. If thepercentage of positive cells for PVRIG, PD-1, PD-1 and PVRL2 is >1% forall 4 receptors, then the patient is treated with antibodies to PVRIGand PD-1 as outlined herein.

In one embodiment, a biopsy is taken from a tumor from a patient withcancer, and dissociated as is known in the art for FACS analysis. Thecells are stained with labeled antibodies to (1) PVRIG (generally usingCHA.7.518.1H4(S241P), for example, although any outlined inWO2016/134333 (specifically including any that bind, even if they don'tblock) or WO2017/041004) can be used); (2) TIGIT (for example using anydescribed herein or others in the art such as MBSA43); (3) PVR (forexample using those known in the art such as SKII.4) and (4) PVRL2 (forexample using those known in the art such as TX11); and (5) a relevantisotype control antibody for the antibodies used. FACS is done, and foreach receptor, the percentage of the cells expressing the receptorrelative to the control antibody is calculated. If the percentage ofpositive cells for PVRIG, TIGIT, PVR and PVRL2 is >1% for all 4receptors, then the patient is treated with antibodies to PVRIG andTIGIT. Preferred combinations in this regard are CHA.7.518.1.H4(S241P)and CPA.9.086.

In one embodiment, a biopsy is taken from a tumor from a patient withcancer, and dissociated as is known in the art for FACS analysis. Thecells are stained with labeled antibodies to (1) PVRIG (generally usingCHA.7.518.1H4(S241P), for example, although any outlined inWO2016/134333 (specifically including any that bind, even if they don'tblock) or WO2017/041004) can be used); (2) TIGIT (for example using anydescribed herein or others in the art such as MBSA43); (3) PVR (forexample using those known in the art such as SKII.4) and (4) PVRL2 (forexample using those known in the art such as TX11); (5) PD-1 (forexample using those known in the art including EH12.2H7, Keytruda®,Opdivo®, Cemiplimab, etc.); and (6) a relevant isotype control antibodyfor the antibodies used. FACS is done, and for each receptor, thepercentage of the cells expressing the receptor relative to the controlantibody is calculated. If the percentage of positive cells for PVRIG,TIGIT, PVR, PVRL2 and PD-1 is >1% for all 5 receptors, then the patientis treated with antibodies to PVRIG, TIGIT, and PD-1. Preferredcombinations in this regard are CHA.7.518.1.H4(S241P), CPA.9.086, andEH12.2H7. Other preferred combinations in this regard areCHA.7.518.1.H4(S241P), CPA.9.086, and Keytruda®. Yet other preferredcombinations in this regard are CHA.7.518.1.H4(S241P), CPA.9.086, andOpdivo®.

In one embodiment, a biopsy is taken from a tumor from a patient withcancer, and dissociated as is known in the art for FACS analysis. Thecells are stained with labeled antibodies to (1) PVRIG (generally usingCHA.7.518.1H4(S241P), for example, although any outlined inWO2016/134333 (specifically including any that bind, even if they don'tblock) or WO2017/041004) can be used); (2) TIGIT (for example using anydescribed herein or others in the art such as MBSA43); ((3) PD-L1 (forexample using those known in the art such as BM-1, atezolizumab,avelumab, and durvalumab, outlined herein) and (4) PVR (for exampleusing those known in the art such as SKII.4); (5) PD-1 (for exampleusing those known in the art including EH12.2H7, Keytruda®, Opdivo®,Cemiplimab, etc.); and (6) a relevant isotype control antibody for theantibodies used. FACS is done, and for each receptor, the percentage ofthe cells expressing the receptor relative to the control antibody iscalculated. If the percentage of positive cells for PVRIG, TIGIT, PD-L1,PVR and PD-1 is >1% for all 5 receptors, then the patient is treatedwith antibodies to PVRIG, TIGIT, and PD-1. Preferred combinations inthis regard are CHA.7.518.1.H4(S241P), CPA.9.086, and EH12.2H7. Otherpreferred combinations in this regard are CHA.7.518.1.H4(S241P),CPA.9.086, and Keytruda®. Yet other preferred combinations in thisregard are CHA.7.518.1.H4(S241P), CPA.9.086, and Opdivo®.

3. Combination Therapy of Anti-TIGIT and Anti-PVRIG Antibodies with PD-1Antibodies for Refractory Patients

In some embodiments, the treatment includes a combination of anti-TIGITantibodies, anti-PVRIG antibodies, and anti-PD-1 antibodies fortargeting tumor cells with high PD-L1 expression. In some embodiments,the treatment includes a combination of anti-TIGIT antibodies,anti-PVRIG antibodies, and anti-PD-1 antibodies for use in a patientwhose tumors express PD-L1. In some embodiments, the treatment includesa combination of anti-TIGIT antibodies, anti-PVRIG antibodies, andanti-PD-1 antibodies for use in a cancer patient whose tumor expressesPD-L1 and/or who is refractory to anti-PD-1 therapeutics. In someembodiments, the treatment includes a combination of anti-TIGITantibodies, anti-PVRIG antibodies, and anti-PD-1 antibodies for use in acancer patient whose tumor expresses PD-L1 and who is refractory toanti-PD-1 therapeutics.

Accordingly, in one embodiment, antibodies containing the CDR sets fromthe anti-TIGIT antibody CPA.9.083 are combined with antibodiescontaining the CDR sets from the anti-PVRIG antibody CHA.7.518.1 andpembrolizumab for use in a cancer patient whose tumor expresses PD-L1and/or who is refractory to anti-PD-1 therapeutics. In a particularembodiment, CPA.9.083.H4(S241P) is combined with CHA.7.518.1.H4(S241P)and pembrolizumab for use in a cancer patient whose tumor expressesPD-L1 and/or who is refractory to anti-PD-1 therapeutics.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CPA.9.083 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.538.1.2 and pembrolizumafor use in a cancer patient whose tumor expresses PD-L1 and/or who isrefractory to anti-PD-1 therapeutics b. In a particular embodiment,CPA.9.083.H4(S241P) is combined with CHA.7.538.1.2.H4(S241P) andpembrolizumab for use in a cancer patient whose tumor expresses PD-L1and/or who is refractory to anti-PD-1 therapeutics.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CPA.9.086 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.518.1 and pembrolizumabfor use in a cancer patient whose tumor expresses PD-L1 and/or who isrefractory to anti-PD-1 therapeutics. In a particular embodiment,CPA.9.086.H4(S241P is combined with CHA.7.518.1.H4(S241P) andpembrolizumab for use in a cancer patient whose tumor expresses PD-L1and/or who is refractory to anti-PD-1 therapeutics.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CPA.9.086 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.538.1.2 andpembrolizumab for use in a cancer patient whose tumor expresses PD-L1and/or who is refractory to anti-PD-1 therapeutics. In a particularembodiment, CPA.9.086.H4(S241P) is combined with CHA.7.538.1.2.H4(S241P)and pembrolizumab for use in a cancer patient whose tumor expressesPD-L1 and/or who is refractory to anti-PD-1 therapeutics.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CHA.9.547.7 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.518.1 and pembrolizumabfor use in a cancer patient whose tumor expresses PD-L1 and/or who isrefractory to anti-PD-1 therapeutics. In a particular embodiment,CHA.9.547.7H4(S241P) is combined with CHA.7.518.1.H4(S241P) andpembrolizumab for use in a cancer patient whose tumor expresses PD-L1and/or who is refractory to anti-PD-1 therapeutics.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CHA.9.547.7 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.538.1.2 andpembrolizumab for use in a cancer patient whose tumor expresses PD-L1and/or who is refractory to anti-PD-1 therapeutics. In a particularembodiment, CHA.9.547.7.H4(S241P) is combined withCHA.7.538.1.2.H4(S241P) and pembrolizumab for use in a cancer patientwhose tumor expresses PD-L1 and/or who is refractory to anti-PD-1therapeutics.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CHA.9.547.13 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.518.1 and pembrolizumabfor use in a cancer patient whose tumor expresses PD-L1 and/or who isrefractory to anti-PD-1 therapeutics. In a particular embodiment,CHA.9.547.13.H4(S241P) is combined with CHA.7.518.1.H4(S241P) andpembrolizumab for use in a cancer patient whose tumor expresses PD-L1and/or who is refractory to anti-PD-1 therapeutics.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CHA.9.547.13 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.538.1.2 andpembrolizumab for use in a cancer patient whose tumor expresses PD-L1and/or who is refractory to anti-PD-1 therapeutics. In a particularembodiment, CHA.9.547.13.H4(S241P) is combined withCHA.7.538.1.2.H4(S241P) and pembrolizumab for use in a cancer patientwhose tumor expresses PD-L1 and/or who is refractory to anti-PD-1therapeutics.

Accordingly, in one embodiment, antibodies containing the CDR sets fromthe anti-TIGIT antibody CPA.9.083 are combined with antibodiescontaining the CDR sets from the anti-PVRIG antibody CHA.7.518.1 andnivolumab for use in a cancer patient whose tumor expresses PD-L1 and/orwho is refractory to anti-PD-1 therapeutics. In a particular embodiment,CPA.9.083.H4(S241P) is combined with CHA.7.518.1.H4(S241P) and nivolumabfor use in a cancer patient whose tumor expresses PD-L1 and/or who isrefractory to anti-PD-1 therapeutics.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CPA.9.083 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.538.1.2 and nivolumabfor use in a cancer patient whose tumor expresses PD-L1 and/or who isrefractory to anti-PD-1 therapeutics. In a particular embodiment,CPA.9.083.H4(S241P) is combined with CHA.7.538.1.2.H4(S241P) andnivolumab for use in a cancer patient whose tumor expresses PD-L1 and/orwho is refractory to anti-PD-1 therapeutics.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CPA.9.086 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.518.1 and nivolumab foruse in a cancer patient whose tumor expresses PD-L1 and/or who isrefractory to anti-PD-1 therapeutics. In a particular embodiment,CPA.9.086.H4(S241P is combined with CHA.7.518.1.H4(S241P) and nivolumabfor use in a cancer patient whose tumor expresses PD-L1 and/or who isrefractory to anti-PD-1 therapeutics.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CPA.9.086 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.538.1.2 and nivolumabfor use in a cancer patient whose tumor expresses PD-L1 and/or who isrefractory to anti-PD-1 therapeutics. In a particular embodiment,CPA.9.086.H4(S241P) is combined with CHA.7.538.1.2.H4(S241P) andnivolumab for use in a cancer patient whose tumor expresses PD-L1 and/orwho is refractory to anti-PD-1 therapeutics.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CHA.9.547.7 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.518.1 and nivolumab foruse in a cancer patient whose tumor expresses PD-L1 and/or who isrefractory to anti-PD-1 therapeutics. In a particular embodiment,CHA.9.547.7H4(S241P) is combined with CHA.7.518.1.H4(S241P) andnivolumab for use in a cancer patient whose tumor expresses PD-L1 and/orwho is refractory to anti-PD-1 therapeutics.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CHA.9.547.7 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.538.1.2 and nivolumabfor use in a cancer patient whose tumor expresses PD-L1 and/or who isrefractory to anti-PD-1 therapeutics. In a particular embodiment,CHA.9.547.7.H4(S241P) is combined with CHA.7.538.1.2.H4(S241P) andnivolumab for use in a cancer patient whose tumor expresses PD-L1 and/orwho is refractory to anti-PD-1 therapeutics.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CHA.9.547.13 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.518.1 and nivolumab foruse in a cancer patient whose tumor expresses PD-L1 and/or who isrefractory to anti-PD-1 therapeutics. In a particular embodiment,CHA.9.547.13.H4(S241P) is combined with CHA.7.518.1.H4(S241P) andnivolumab for use in a cancer patient whose tumor expresses PD-L1 and/orwho is refractory to anti-PD-1 therapeutics.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CHA.9.547.13 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.538.1.2 and nivolumabfor use in a cancer patient whose tumor expresses PD-L1 and/or who isrefractory to anti-PD-1 therapeutics. In a particular embodiment,CHA.9.547.13.H4(S241P) is combined with CHA.7.538.1.2.H4(S241P) andnivolumab for use in a cancer patient whose tumor expresses PD-L1 and/orwho is refractory to anti-PD-1 therapeutics.

In some embodiments, the anti-TIGIT antibody is an antibody chosen fromany anti-TIGIT antibody described herein, including any of thosedescribed in FIG. 3 . In some embodiments, the anti-PVRIG antibody is anantibody chosen from any anti-PVRIG antibody described herein, includingany of those described in FIG. 5 and/or FIG. 63 . In some embodiments,the anti-PD-1 antibody is an antibody chosen from any anti-PD-1 antibodydescribed herein, including any of those described in FIG. 7 .

4. Assessment of Treatment

Generally, the antibodies of the invention, alone or in combination(PVRIG with PD-1, TIGIT with PD-1 or TIGIT with PVRIG, and/or PVRIG withboth TIGIT and PD-1) are administered to patients with cancer, andefficacy is assessed, in a number of ways as described herein. Thus,while standard assays of efficacy can be run, such as cancer load, sizeof tumor, evaluation of presence or extent of metastasis, etc.,immuno-oncology treatments can be assessed on the basis of immune statusevaluations as well. This can be done in a number of ways, includingboth in vitro and in vivo assays. For example, evaluation of changes inimmune status (e.g. presence of ICOS+CD4+ T cells following ipitreatment) along with “old fashioned” measurements such as tumor burden,size, invasiveness, LN involvement, metastasis, etc. can be done. Thus,any or all of the following can be evaluated: the inhibitory effects ofPVRIG on CD4⁺ T cell activation or proliferation, CD8⁺ T (CTL) cellactivation or proliferation, CD8⁺ T cell-mediated cytotoxic activityand/or CTL mediated cell depletion, NK cell activity and NK mediatedcell depletion, the potentiating effects of PVRIG on Treg celldifferentiation and proliferation and Treg- or myeloid derivedsuppressor cell (MDSC)-mediated immunosuppression or immune tolerance,and/or the effects of PVRIG on proinflammatory cytokine production byimmune cells, e.g., IL-2, IFN-γ or TNF-α production by T or other immunecells.

In some embodiments, assessment of treatment is done by evaluatingimmune cell proliferation, using for example, CFSE dilution method, Ki67intracellular staining of immune effector cells, and 3H-Thymidineincorporation method.

In some embodiments, assessment of treatment is done by evaluating theincrease in gene expression or increased protein levels ofactivation-associated markers, including one or more of: CD25, CD69,CD137, ICOS, PD1, GITR, OX40, and cell degranulation measured by surfaceexpression of CD107A.

In some embodiments, the assessment of treatment is done by assessingthe amount of T cell proliferation in the absence of treatment, forexample prior to administration of the antibodies of the invention. If,after administration, the patient has an increase in T cellproliferation, e.g. a subset of the patient's T cells are proliferating,this is an indication that the T cells were activated.

Similarly, assessment of treatment with the antibodies of the inventioncan be done by measuring the patient's IFN-γ levels prior toadministration and post-administration to assess efficacy of treatment.This may be done within hours or days.

In general, gene expression assays are done as is known in the art. Seefor example Goodkind et al., Computers and Chem. Eng. 29(3):589 (2005),Han et al., Bioinform. Biol. Insights 11/15/15 9(Suppl. 1):29-46, Campoet al., Nod. Pathol. 2013 Jan; 26 suppl. 1:S97-S110, the gene expressionmeasurement techniques of which are expressly incorporated by referenceherein.

In general, protein expression measurements are also similarly done asis known in the art, see for example, Wang et al., Recent Advances inCapillary Electrophoresis-Based Proteomic Techniques for BiomarkerDiscovery, Methods. Mol. Biol. 2013:984:1-12; Taylor et al, BioMed Res.Volume 2014, Article ID 361590, 8 pages, Becerk et al., Mutat. Res 2011Jun. 17:722(2): 171-182, the measurement techniques of which areexpressly incorporated herein by reference.

In some embodiments, assessment of treatment is done by assessingcytotoxic activity measured by target cell viability detection viaestimating numerous cell parameters such as enzyme activity (includingprotease activity), cell membrane permeability, cell adherence, ATPproduction, co-enzyme production, and nucleotide uptake activity.Specific examples of these assays include, but are not limited to,Trypan Blue or PI staining, ⁵¹Cr or ³⁵S release method, LDH activity,MTT and/or WST assays, Calcein-AM assay, Luminescent based assay, andothers.

In some embodiments, assessment of treatment is done by assessing T cellactivity measured by cytokine production, measure either intracellularlyin culture supernatant using cytokines including, but not limited to,IFN-γ, TNF-α, GM-CSF, IL-2, IL-6, IL-4, IL-5, IL-10, and/or IL-13 usingwell known techniques.

Accordingly, assessment of treatment can be done using assays thatevaluate one or more of the following: (i) increases in immune response,(ii) increases in activation of αβ and/or γδ T cells, (iii) increases incytotoxic T cell activity, (iv) increases in NK and/or NKT cellactivity, (v) alleviation of αβ and/or γδ T-cell suppression, (vi)increases in pro-inflammatory cytokine secretion, (vii) increases inIL-2 secretion; (viii) increases in interferon-y production, (ix)increases in Th1 response, (x) decreases in Th2 response, (xi) decreasesor eliminates cell number and/or activity of at least one of regulatoryT cells (Tregs).

Assays to Measure Efficacy

In some embodiments, T cell activation is assessed using a MixedLymphocyte Reaction (MLR) assay as is described in the Examples. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in immune response as measured for an example byphosphorylation or de-phosphorylation of different factors, or bymeasuring other post translational modifications. An increase inactivity indicates immunostimulatory activity. Appropriate increases inactivity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in activation of αβ and/or γδ T cells as measured for anexample by cytokine secretion or by proliferation or by changes inexpression of activation markers like for an example CD137, CD107a, PD1,etc. An increase in activity indicates immunostimulatory activity.Appropriate increases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in cytotoxic T cell activity as measured for an example bydirect killing of target cells like for an example cancer cells or bycytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in NK and/or NKT cell activity as measured for an example bydirect killing of target cells like for an example cancer cells or bycytokine secretion or by changes in expression of activation markerslike for an example CD107a, etc. An increase in activity indicatesimmunostimulatory activity. Appropriate increases in activity areoutlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in αβ and/or γδ T-cell suppression, as measured for an exampleby cytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in pro-inflammatory cytokine secretion as measured for exampleby ELISA or by Luminex or by Multiplex bead based methods or byintracellular staining and FACS analysis or by Alispot etc. An increasein activity indicates immunostimulatory activity. Appropriate increasesin activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in IL-2 secretion as measured for example by ELISA or byLuminex or by Multiplex bead based methods or by intracellular stainingand FACS analysis or by Alispot etc. An increase in activity indicatesimmunostimulatory activity. Appropriate increases in activity areoutlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in interferon-γ production as measured for example by ELISA orby Luminex or by Multiplex bead based methods or by intracellularstaining and FACS analysis or by Alispot etc. An increase in activityindicates immunostimulatory activity. Appropriate increases in activityare outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in Th1 response as measured for an example by cytokinesecretion or by changes in expression of activation markers. An increasein activity indicates immunostimulatory activity. Appropriate increasesin activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in Th2 response as measured for an example by cytokinesecretion or by changes in expression of activation markers. An increasein activity indicates immunostimulatory activity. Appropriate increasesin activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases cell number and/or activity of at least one of regulatory Tcells (Tregs), as measured for example by flow cytometry or by IHC. Adecrease in response indicates immunostimulatory activity. Appropriatedecreases are the same as for increases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in M2 macrophages cell numbers, as measured for example byflow cytometry or by IHC. A decrease in response indicatesimmunostimulatory activity. Appropriate decreases are the same as forincreases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in M2 macrophage pro-tumorigenic activity, as measured for anexample by cytokine secretion or by changes in expression of activationmarkers. A decrease in response indicates immunostimulatory activity.Appropriate decreases are the same as for increases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in N2 neutrophils increase, as measured for example by flowcytometry or by IHC. A decrease in response indicates immunostimulatoryactivity. Appropriate decreases are the same as for increases, outlinedbelow.

In one embodiment, the signaling pathway assay measures increases ordecreases in N2 neutrophils pro-tumorigenic activity, as measured for anexample by cytokine secretion or by changes in expression of activationmarkers. A decrease in response indicates immunostimulatory activity.Appropriate decreases are the same as for increases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in inhibition of T cell activation, as measured for an exampleby cytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in inhibition of CTL activation as measured for an example bydirect killing of target cells like for an example cancer cells or bycytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in αβ and/or γδ T cell exhaustion as measured for an exampleby changes in expression of activation markers. A decrease in responseindicates immunostimulatory activity. Appropriate decreases are the sameas for increases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases αβ and/or γδ T cell response as measured for an example bycytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in stimulation of antigen-specific memory responses asmeasured for an example by cytokine secretion or by proliferation or bychanges in expression of activation markers like for an example CD45RA,CCR7 etc. An increase in activity indicates immunostimulatory activity.Appropriate increases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in apoptosis or lysis of cancer cells as measured for anexample by cytotoxicity assays such as for an example MTT, Cr release,Calcine AM, or by flow cytometry based assays like for an example CFSEdilution or propidium iodide staining etc. An increase in activityindicates immunostimulatory activity. Appropriate increases in activityare outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in stimulation of cytotoxic or cytostatic effect on cancercells. as measured for an example by cytotoxicity assays such as for anexample MTT, Cr release, Calcine AM, or by flow cytometry based assayslike for an example CFSE dilution or propidium iodide staining etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases direct killing of cancer cells as measured for an example bycytotoxicity assays such as for an example MTT, Cr release, Calcine AM,or by flow cytometry based assays like for an example CFSE dilution orpropidium iodide staining etc. An increase in activity indicatesimmunostimulatory activity. Appropriate increases in activity areoutlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases Th17 activity as measured for an example by cytokine secretionor by proliferation or by changes in expression of activation markers.An increase in activity indicates immunostimulatory activity.Appropriate increases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in induction of complement dependent cytotoxicity and/orantibody dependent cell-mediated cytotoxicity, as measured for anexample by cytotoxicity assays such as for an example MTT, Cr release,Calcine AM, or by flow cytometry based assays like for an example CFSEdilution or propidium iodide staining etc. An increase in activityindicates immunostimulatory activity. Appropriate increases in activityare outlined below.

In one embodiment, T cell activation is measured for an example bydirect killing of target cells like for an example cancer cells or bycytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. ForT-cells, increases in proliferation, cell surface markers of activation(e.g. CD25, CD69, CD137, PD1), cytotoxicity (ability to kill targetcells), and cytokine production (e.g. IL-2, IL-4, IL-6, IFNγ, TNF-α,IL-10, IL-17A) would be indicative of immune modulation that would beconsistent with enhanced killing of cancer cells.

In one embodiment, NK cell activation is measured for example by directkilling of target cells like for an example cancer cells or by cytokinesecretion or by changes in expression of activation markers like for anexample CD107a, etc. For NK cells, increases in proliferation,cytotoxicity (ability to kill target cells and increases CD107a,granzyme, and perforin expression), cytokine production (e.g. IFNy andTNF), and cell surface receptor expression (e.g. CD25) would beindicative of immune modulation that would be consistent with enhancedkilling of cancer cells.

In one embodiment, γδ T cell activation is measured for example bycytokine secretion or by proliferation or by changes in expression ofactivation markers.

In one embodiment, Th1 cell activation is measured for example bycytokine secretion or by changes in expression of activation markers.

Appropriate increases in activity or response (or decreases, asappropriate as outlined above), are increases of at least about 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98 to 99% percent overthe signal in either a reference sample or in control samples, forexample test samples that do not contain an anti-PVRIG antibody of theinvention. Specific increases in activity are depicted in the attachedfigures. For example, with regard to increases in T cell proliferation,CHA.7.518.1.H4(S241P) shows an increase of about 60% andCHA.7.538.1.2.H4(S241P) shows an increase of 47%; relevant increases areshown in either T cell proliferation or IFN-γ of from about 10 to 70%with from about 20 to 60% also finding use.

Similarly, increases of at least one-, two-, three-, four- or five-foldas compared to reference or control samples show efficacy.

X. EXAMPLES Example 1 Functional Assays

The purpose of this study was to characterize the functional activityCHA.7.518.1.H4(S241P) on human T cell function either alone or incombination with an anti-TIGIT and/or anti-PD-1 antibody in primary invitro cell-based assays. We demonstrate that CHA.7.518.1.H4(S241P)enhanced cytokine production of viral antigen-specific CD8 T cellsutilized as a model surrogate antigen to study CD8 T cell responses.Combination of CHA.7.518.1.H4(S241P) with anti-TIGIT antibody lead to anadditive or, in some conditions, synergistic increase in T cellfunction. We also performed a triple combination ofCHA.7.518.1.H4(S241P), anti-TIGIT, and anti-PD-1 and observed thegreatest increase in T cell function co-cultured with PD-L1hi targettumor cells using the triple combination as compared to the doublecombination or the individual antibody. In a co-culture with PD-Lilotarget tumor cells, the triple combination of CHA.7.518.1.H4(S241P),anti-TIGIT, anti-PD-1 did not further enhance T cell function comparedto CHA.7.518.1.H4(S241P) and anti-TIGIT double combination, suggestingthat CHA.7.518.1.H4(S241P) and anti-TIGIT treatment may be effective inpatients who have low or negative PD-L1 expression. Taken together, wedemonstrate an effect of CHA.7.518.1.H4(S241P) in enhancing human CD8+ Tcell function, either individually or in combination with anti-TIGIT oranti-PD-1. This report describes the characterization ofCHA.7.518.1.H4(S241P), a fully humanized IgG4 anti-PVRIG antibody, incell based assays. CHA.7.518.1.H4(S241P) binds to PVRIG with highaffinity and specificity, and blocks the interaction of PVRIG withPVRL2. In order to understand the effects of CHA.7.518.1.H4(S241P) on Tcell function, we examined the effect of CHA.7.518.1.H4(S241P) oncytokines production in an in vitro assay. This assay was designed basedon the 2 signal hypothesis of T cell activation: signal 1 comes from theactivation of the T cell receptor; signal 2 are immunomodulatoryreceptors that help to enhance or inhibit the T cell responses. Thedesign of these assays consist of a co-culture of human T cells with atarget cell line pulsed with antigen peptide derived from a viralantigen (CMV). This signal provides “signal 1” of T cell activationthrough the T cell receptor. These target cell lines express endogenousPVRL2, and in this context PVRL2 provides “signal 2” to the T cell.

CMV: Tumor Cell Line Assay

CMVpp65-reactive T cells were expanded by thawing CMV-reactive donorsaccording to CTL “Thawing Cryopreserved PBMC” protocol and 2e6 cells/mlwere resuspended in medium (Gibco) supplemented with 1% glutamax(Gibco), 1% NEAA, penicillin/streptomycin (Gibco), 10% human AB serum(Corning), 1 ug/ml CMV peptide, 2 ng/ml IL-2 (R&D), and 10 ng/ml IL-7(R&D). PBMCs were cultured for eight days with IL-2 and IL-7 replenishedat day three and day six. At Day 8, cells were harvested, and replatedin low dose IL-2 (100 U/ml) at 2e6/ml in complete RPMI media for 5 days.At day nine, cells are phenotyped for CD8+T cell purity and CMV tetramerreactivity. Cells were stained with 0.25 μL of CD3(clone:OKT)-allophvcocyanin seven (APC-Cy7;Biolegend), 0.25 ul of CD8 (clone:HiT8a)-Alexafluor 488 (AF488; Biolegend.), 0.125 μL of CD14 (clone:HCD14), 0.5 ul of CD19 (clone:HIBCD14), 0.5 ul of CD56(cione:HCD56)-peridinin chlorophyll protein (PerCP;Biolegend), 1.25 ulμL of TIGIT (clone:MBSA43)-allophycocyanin (APC;e-Bioscience) or 1.25 μLof IgG4(In house)-isotype control (APC:Biolegend), 1.25 ul ofCHA.7.518.1.H4(S241P)-allophycocyanin (In house), or 1.25 μL of IgG4-APCisotype control (In house) and 0.5 ul of PD-1 (clone:EH12.2H7)-Brilliant Violet 421 (BV421;Biolegend) or 1.25 ul of IgG1(clone: MOPC21)-Brilliant Violet 421 (BV421;Biolegend). To assess thefrequency of tetramer-reactive CD8⁺ T cells, unlabeled PBMCs werestained after cultivation) with 10 μL of iTAg Tetramer—HLA-A*02:01 CMVpp65 (NLVPMVATV)-phycoerythrin (PE, MSL-BION) for 30 min at roomtemperature. Cells were washed with PBS/BSA/azide solution andresuspended in buffer). Data was acquired using a Fortessa, and analyzedusing FlowJo (Treestar) and Prism (Graphpad) software.

The target cells used in the co-culture assay were Panc.05.4 and Coio205cell lines (ATCC). These cell lines were stained with 1.25 ul ofPVR(SKII.4)-phycoerythrin (PE,Biolegend), 1.25 ul of PVRL2(TX31)-peridinin chlorophyll protein (PerCP5.5;Biolegend), 2.5 ulPD-L1(29E.2A3)-Brilliant Violet 785 (BV785;Biolegend) and 1.25 ul ofHLA-A2 (BB7.2)-allophycocyanin (APC; Biolegend) expression. 1.25 ul ofthe corresponding isotype for each flurophore was also assessed(MOPC-21).

To setup the co-culture, tumor cell lines were harvested from cultureand tumor cell lines pulsed with CMV peptide (Anaspec) for 1 hour at 37°C. with periodic mixing. After the incubation, the target cells werethoroughly washed, counted, and re-suspended in complete RPMI medium.The assays were set up with a 1:1 ratio of T cells (100,000) to targetcells (100,000). The target cells, T cells and 10 ug/ml of each antibodytreatment were added together in a 96-well U bottom plate (Costar), andincubated for 24 hrs at 37° C. The antibody treatments include,CHA.7.518.1.H4(S241P) hIgG4, anti-TIGIT hIgG4 (Benchmark 26, Compugen),Anti-PD-1 hIgG4 (Benchmark 3, Compugen) and a human IgG4 isotype control(Compugen). In order to match the total antibody concentration acrossall individual and combination groups, additional human IgG4 isotypecontrol was added in to single or double combination conditions to afinal total antibody concentration of 30 ug/ml. After the 24 hrincubation period, co-culture supernatants were analyzed for secretedcytokines, including IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A,IL-17F, IL-21, IL-22, TNF-α, and/or IFN-γ, with the cytometric beadarray (CBA) human Th1/Th2/Th17 cytokine kit (BD Biosciences), or withthe LEGENDplex™ Human Th cytokine kit (BioLegend). Data was acquiredusing a Fortessa, and analyzed using FlowJo (Treestar) and Prism(Graphpad) software

1. Results and Discussion

a. CMV T Cell Assay: CMVpp65 Reactive T Cells Express PVRIG, TIGIT, andPD1

Human cytomegalovirus (CMV) is a widespread persistent β-herpesvirusthat infects a high percentage of the population, with slightly lowerseroprevalance in Western Europe and the United States (Cannon M J etal. 2010). The immune system of patients with chronic viral infectionsor cancer is often impaired in function and is unable to mount aneffective response against the virus or to recognize and eliminatemalignant cells. In these patients, expression of inhibitory receptorsincrease and this was found to be associated with T-cell dysfunction.Thus, the upregulation of negative checkpoints receptors may serve aspotential targets for the reversal of T cell exhaustion. CD8 T cellsspecific to CMV pp65 protein have been well characterized and these CMVspecific T cells can be used to study the role of modulatory receptorson T cells.

Stimulation of HLA-A2+ donor PBMCs using CMV pp65 peptide, IL-2 and IL-7resulted in a strong expansion of CMV pp65-specific T cells to puritiesranging from 50-90% as determined by tetramer staining. FIG. 8A showsthe percentage of CMV pp65 specific T cells from several donors afterexpansion. The surface expression of PVRIG, TIGIT and PD-1 on T cellswas assessed from CMV+ donors, and compared to respective isotype forreceptor expression by flow cytometry. CMV pp65 specific T cellsexpressed PVRIG (median gMFI ratio: 7), TIGIT (median gMFI ratio: 37),and PD1 (median gMFI ratio: 2) on day 9 of activation (FIG. 8B).

We further assessed the kinetics of expression of PVRIG, TIGIT, and PD1relative to the expansion of CMV pp65 specific T cells in a time course.For each donor, the frequency of CMVpp65 reactive CD8+ T cells areplotted over time (FIG. 9A). A significant expansion in the frequency ofCMVpp65 reactive T cells (range: 50-97%) was observed in all donors,with donor 198 initially expanding at day three (Day 3 CMV+percentage:85.7%). However, donor 198 had a loss of CMV tetramer expression at day6 (Day 6 CMV+percentage: 50.4%). PVRIG, TIGIT and PD-1 expression ofCMV-specific CD8⁺ T cells was assessed by flow cytometry over thetwelve-day time course. In donor 4, donor 198 and donor 210, TIGITexpression among CMVpp65 specific CD8⁺ T cells increased during thetwelve-day expansion period (mean gMFIr expression of three donors TIGITexpression of three donors, Day 0 gMFIr: 1.2, Day 12 gMFIr: 47) (FIG.9B). PVRIG expression of CMV+T cells also increased (mean gMFI PVRIG ofthree donors, Day 0 gMFIr: 0.92, Day 12 gMFIr: 8.6) (FIG. 6C). PD-1expression was also assessed, and we observed minimal induction ofexpression (mean gMFIr PD-1, Day 0 gMFI: 0.93, Day 12 gMFI: 2) (FIG.9C).

b. CHA.7.518.1.H4(S241P), Anti-TIGIT, and Anti-PD1 Antibodies EnhancedIFN-γ Secretion

With the rationale that the upregulation of TIGIT, PVRIG, and PD-1expression by CD8 CMV cells correlates with T cell dysfunction, we aimedto evaluate the effects of PVRIG, TIGIT, and PD-1 blockade on thecapacity for pro-inflammatory cytokine production. CMVpp65 reactive Tcells from 2 donors were co-cultured with CMV peptide loaded PD-L1hi(Panc04.05) and PD-L1lo (Colo205) tumor cell lines prior to flowcytometric analysis of cytokine production (FIG. 10 ).

In the Panc.04.05 (PD-L1hi) co-culture, we observed that anti-TIGITsingle blockade increased IFN-y production compared with IgG controlmAbs, whereas CHA.7.518.1.H4(S241P) or anti-PD-1 had minimal effect(FIG. 11 ). Dual anti-TIGIT and CHA.7.518.1.H4(S241P) blockadesynergistically and consistently increased the cytokine production ofCD8+ T cells as compared with CHA.7.518.1.H4(S241P) or anti-TIGIT singleblockade alone. In donor 4, a further increase in IFN-γ was observedwith a triple combination of CHA.7.518.1.H4(S241P), anti-TIGIT, andanti-PD-1, suggesting that when PD-L1, PVR, and PVRL2 are expressed athigh levels on tumor cells, the largest increase in T cell activation isachieved with a triple combination. In Colo205 (PD-L11o) co-cultures,anti-TIGIT blockade alone increased IFN-y secretion whereasCHA.7.518.1.H4(S241P) or anti-PD1 antibody had minimal effect, similarto the results with Panc.04.05 co-culture. Also similar to thePanc.04.05 co-culture, dual blockade of anti-TIGIT andCHA.7.518.1.H4(S241P) also synergistically increased IFN-y as comparedanti-TIGIT, anti-PD-1, or CHA.7.518.1.H4(S241P) alone, and to a greatermagnitude of either CHA.7.518.1.H4(S241P) or anti-TIGIT in combinationwith anti-PD-1. In contrast to Panc.04.05 (PD-L1hi), the triplecombination condition for Donor 4 was not better than the dualcombination of CHA.7.518.1.H4(S241P) and anti-TIGIT in the Colo205co-culture (PD-L1lo), suggesting when PVR and PVRL2 are expressed athigh levels and PD-L1 at a low level on tumor cells, double combinationCHA.7.518.1.H4(S241P) and anti-TIGIT led to the greatest increase ofIFN-γ expression. These findings demonstrate that TIGIT and PVRIGblockade were sufficient to enhance CD8+ T cell responses in PD-L1lotumors and that a triple combination led to the greatest increase in Tcell activation in PD-L1hi tumors.

c. SUMMARY

Human anti-CMV T cell responses are utilized as an in-vitroantigen-specific method to assess checkpoint inhibitor antibodyfunctional capability. We observed that co-blockade of TIGIT andCHA.7.518.1.H4(S241P) lead to a greater restoration of T cell functioncompared with a single antibody blockade, suggesting that disrupting theTIGIT and PVRIG pathway may be more important that disrupting the PD1pathway in CD8-tumor cell co-cultures. Furthermore, we observed thattriple blockade using antibodies to PD-1, TIGIT, and PVRIG can result inthe greatest increase in IFN-γ when PD-L1 positive tumor cells or immunecells are >1%, which is equivalent to PD-L1 high expression levels.These findings demonstrate that TIGIT and PVRIG blockade were sufficientto enhance CD8+ T cell responses in PD-L1lo tumors and that a triplecombination led to the greatest increase in T cell activation in PD-L1hitumors.

The present invention provides methods comprising: a) providing a cellpopulation from a tumor sample from a patient; b) staining saidpopulation with labeled antibodies that bind: i) TIGIT protein; ii)PVRIG protein; iii) PVR protein; iv) PD-1 protein; v) PD-L1 protein; vi)PVRL2; and vii) a relevant isotype control for the antibodies in i)-vi);c) running fluorescence activated cell sorting (FACS); d) for each ofTIGIT, PVRIG, PVR, PD-1, PVRL2 and PD-L1, determining the percentage ofcells in said population that express the protein relative to saidisotype control antibody; wherein if the percentage of positive cellsis >1% for either TIGIT or PVR, and for either PVRIG or PVRL2, and foreither PD-1 or PD-L1, proceeding to step e); and e) administeringantibodies to TIGIT, PVRIG, and PD-1 to said patient.

The present invention provides a method comprising: a) providing a cellpopulation from a tumor sample from a patient; b) staining saidpopulation with labeled antibodies that bind: i) TIGIT protein; ii)PVRIG protein; iii) PVR protein; iv) PD-1 protein; v) PD-L1 protein; vi)PVRL2; and vii) a relevant isotype control for the antibodies in i)-vi);c) running fluorescence activated cell sorting (FACS); d) for each ofTIGIT, PVRIG, PVR, PD-1, PVRL2 and PD-L1, determining the percentage ofcells in said population that express the protein relative to saidisotype control antibody; wherein if the percentage of positive cellsis >1% for either TIGIT or PVR, and for either PVRIG or PVRL2, and foreither PD-1 or PD-L1, proceeding to step e); and e) administeringantibodies to TIGIT, PVRIG, and PD-1 to said patient.

The present invention provides a method comprising: a) providing a cellpopulation from a tumor sample from a patient; b) staining saidpopulation with labeled antibodies that bind: i) TIGIT protein; and ii)PVR protein; and iii) a relevant isotype control for the antibodies ini)-ii); c) running fluorescence activated cell sorting (FACS); d) foreach of TIGIT and PVR, determining the percentage of cells in saidpopulation that express the protein relative to said isotype controlantibody; wherein if the percentage of positive cells is >1% for eitherTIGIT or PVR, proceeding to step e); and e) administering antibodies toTIGIT, PVRIG, and PD-1 to said patient.

The present invention provides a method comprising: a) providing a cellpopulation from a tumor sample from a patient; b) staining saidpopulation with labeled antibodies that bind: i) PVRIG protein; and ii)PVRL2 protein; and iii) a relevant isotype control for the antibodies ini)-ii); c) running fluorescence activated cell sorting (FACS); d) foreach of PVRIG and PVRL2, determining the percentage of cells in saidpopulation that express the protein relative to said isotype controlantibody; wherein if the percentage of positive cells is >1% for eitherPVRIG or PVRL2, proceeding to step e); and e) administering antibodiesto TIGIT, PVRIG, and PD-1 to said patient.

The present invention provides a method comprising: a) providing a cellpopulation from a tumor sample from a patient; b) staining saidpopulation with labeled antibodies that bind: i) PD-1 protein; and ii)PD-L1 protein; and iii) a relevant isotype control for the antibodies ini)-ii); c) running fluorescence activated cell sorting (FACS); d) foreach of PD-1 and PD-L1, determining the percentage of cells in saidpopulation that express the protein relative to said isotype controlantibody; wherein if the percentage of positive cells is >1% for eitherPD-1 or PD-L1, proceeding to step e); and e) administering antibodies toTIGIT, PVRIG, and PD-1 to said patient.

Example 2 Expression of PVRIG and PVRL2 IN Human Cancer and NormalAdjacent Tissues

The purpose of this study was to examine the expression of PVRIG andPVRL2 in human tumor and normal adjacent samples. PVRIG was observed tobe expressed highest on CD8+ T cells, followed by NK cells, CD4-CD8- Tcells, and by CD4+ T cells. No expression was observed on monocytes,mDCs, pDCs, or tumor cells. Of the tumor types examined, endometrial,lung and kidney tumors expressed the highest levels of PVRIG onlymphocytes. A comparison of PVRIG expression on CD4+ and CD8+ T cellsfrom normal adjacent tissues compared to tumor tissues from the samepatient showed a significant increase in PVRIG in tumor tissues. Acorrelation analysis of the magnitude of PVRIG expression with themagnitude of TIGIT or PD-1 expression showed a positive and significantcorrelation on CD4 and CD8 T cells. In addition, a co-expression singlecell analysis of PD-1, TIGIT, and PVRIG showed that PVRIG isco-expressed with PD-1 and TIGIT on a subset of cells. These datasupport the conclusion that combination blockade of PVRIG with TIGITand/or PD-1 will lead to increased T cells responses. The ligand forPVRIG, PVRL2, was expressed on myeloid cells (monocytes, mDCs, pDCs) andon CD45-non-immune cells from multiple tumors, likely composed of tumorepithelium and stromal cells. A comparison of PVRL2 on cells derivedfrom normal adjacent vs tumor tissue showed significant increase inexpression of PVRL2 on monocytes and CD45- non-immune cells. Acorrelation analysis of the magnitude of PVRL2 expression with themagnitude of PD-L1 expression showed a positive and significantcorrelation on CD45- non-immune cells and monocytes. In these samples,we also assessed the co-expression of PVRIG and PVRL2 in the same sampleto understand which tumor type has high co-expression of both receptorand ligands. Of the tumor types examined, we observed high expression ofboth PVRIG and PVRL2 in the majority of endometrial samples, kidneysamples, and lung tumor samples. In summary, these data demonstrate thatPVRIG and PVRL2 are expressed on leukocytes and tumor cells from thetumor microenvironment and suggest that this pathway can be exploited toregulate anti-tumor responses.

We examined the expression of PVRIG and PVRL2 using flow cytometry oncells from dissociated human tumors and matched normal adjacent tissuesfrom multiple different tissues. The expression of immune regulators inthe tumor can be used to help predict which tumor types or patients canbe most responsive to a specific therapy.

Healthy human peripheral blood mononuclear cells (PBMC) donors wereobtained from the Stanford Blood Bank. Buffy coats or LRS products werediluted 1:1 in 1×PBS+2% FBS and PBMCs were isolated by Ficoll-Paquegradient (Sigma). Purified PBMCs were washed 2× with PBS+2% FBS andbanked in liquid nitrogen. Tumor and normal adjacent tissue (NAT)samples were provided by the Cooperative Human Tissue Network, aNational Cancer Institute supported resource. The tumor type wasdetermined based on reviewing the pathology report for each sample. Thenumber of samples per tumor type where we examined PVRIG and PVRL2expression is reported below.

Colon, Rectal, Endometrium Target Breast Stomach & Uterine Head&NeckKidney Lung Prostate Ovary PVRIG 4 15 24 4 14 8 9 5 PVPL2 6 21 28 5 1611 12 10

1. Tumor Dissociation Protocol

Tumor and NAT samples were cut into small pieces with a scalpel andtransferred to GentleMACs™ C tubes (Miltenyi Biotec) containing anenzyme mix. Samples were dissociated on GentleMACs (Miltenyi Biotec) asper the manufacturer's protocol. After dissociation, cells were filteredthrough a 100 μm filter prior to FACS staining.

2. Antibodies and Reagents:

To identify immune and non-immune cell populations, the followingantibodies were at the manufacturer's recommended concentrations:

TABLE 2 The antibodies used to identify specific cell subsets is shown.Antibody Flourophore Clone Vendor Cat CD45 Alexa Fluor 700 HI30BioLegend 304024 CD3 APC Cy7 OKT3 BioLegend 317342 CD8 BV 785 RPA-T8BioLegend 301046 CD33 BV711 WM53 BioLegend 303424 CD25 BV 650 BC96BioLegend 302634 CD127 BV 605 A019D5 BioLegend 351334 CD14 BUV395 MoP9BD Pharmingen 563562 CD4 BUV496 SK3 BD Pharmingen 564651 CD56 PE DazzleHCD56 BioLegend 318348

The following antibodies were used at 5 ug/ml in the isotype controlcocktail.

TABLE 3 The antibodies used as isotype controls for the targets ofinterest is shown. Antibody Flourophore Clone Vendor Cat mlgG1 AF647 inhouse Compugen In-house hlgG4 PE in house Compugen In-house mlgG1 BV421MOPC21 BioLegend 400158 mlgG1 PerCP Cy5-5 MOPC21 BioLegend 400150 mlgG1PE Cy7 MOPC21 BioLegend 400126 mlgG1 FITC MOPC21 BioLegend 400110

The following cocktail was used to stain the targets of interest at 5ug/ml:

TABLE 4 The antibodies used to analyze the targets of interest are shownAntibody Flourophore Clone Vendor Cat PDL1 AF647 BM1 (M1) CompugenIn-house PVRIG PE 518 (H4) BioLegend  93930 PD1 BV421 EH12.2H7 BioLegend329920 (M1) PVR PerCP Cy5-5 SKII.4 (M1) BioLegend 337612 PVRL2 PE Cy7TX31 (M1) BioLegend 337414 TIGIT FITC MBSA43 (M1) eBioscience 11-9500-42

All isotype control antibodies and target antibodies were used at 5ug/ml final concentration.

3. FACS Staining

1×10⁶ cells of PBMCs or dissociated tumor cells were seed into a 96-wellV-bottomed plate for staining. Samples were first stained with Aqua LiveDead (Thermo Scientific) to distinguish live cells from dead cells andwith a cocktail of anti-CD16 (Biolegend), anti-CD32 (Thermo Scientific),anti-CD64 (Biolegend) Abs to block Fc receptors. Samples were washedtwice with FACS buffer and stained with a relevant isotype control forthe antibodies for the antibodies used or a target antibody cocktaildescribed in the “Antibodies and Reagents” section. All staining wasdone for 30 minutes at 4C. Samples were then washed twice and acquiredon the BD Fortessa flow cytometer. Analysis was done using FlowJo,gating on specific populations as specified in Table 1, above (all gatedon live cells).

From each population with at least 100 cells, MFI values were exportedand a MFI ratio (MFIr) calculated by dividing the MFI of a target by theMFI of the relevant isotype control. MFIr value greater than one denotespositive expression detected.

4. Results and Discussion a. PVRIG is expressed on TILS from multipletumor types and is co-expressed with TIGIT and PD1

To examine the expression of PVRIG on cells derived from tumors by flowcytometry, tumors were dissociated and stained for immune cell lineagemarkers to identify immune and non-immune cell subsets and for PVRIG,TIGIT, PD1, PVRL2, and PVR to examine expression of these targets onthese subsets. Expression of PVRIG was detected on CD4⁺ T cells, CD8⁺ Tcells, CD4⁻CD8⁻ T cells, and NK cells from breast, colon/rectal/stomach,endometrium, head & neck, lung, kidney, prostate, and ovarian tumors(FIG. 12A). Across all tumor tissues examined, PVRIG was expressed, fromhighest to lowest, on CD8⁺ T cells, NK Cells, CD4⁻CD8⁻T cells, and CD4⁺T cells. No PVRIG expression was detected on monocytes, mDCs, pDCs, ornon-immune cells (FIG. 1B). As CD8⁺ T cells and NK cells are known to beimportant cytotoxic lymphocytes within the immune system, this suggeststhe PVRIG can directly modulate the activity of these cytotoxiclymphocytes.

We next examined the expression of magnitude of PVRIG in relation to themagnitude of TIGIT and PD-1 on tumor infiltrating T cells. For thisanalysis, we focused the analysis on endometrial samples because we hadsufficient number of samples with which to perform the correlationanalysis. PVRIG significantly and directly correlated with TIGIT and PD1expression on both CD4 and CD8 T cells, suggesting that these moleculesare co-regulated within the TME (FIG. 13 ).

We further examined co-expression of PVRIG, TIGIT, and PD1 on a singlecell basis on CD8 T cells. Co-expression of PVRIG with PD-1 and withTIGIT was observed on a representative lung and kidney cancer (FIG. 14).

b. PVRIG Expression is Significantly Enhanced on T Cells from the TumorVersus Normal Adjacent Tissue

For a subset of colon/rectal/stomach, endometrium, kidney, lung orovarian tumors, we were able to obtain matched tumor and normal adjacenttumor (NAT) samples from the same donor. Using these matched samples, wecompared the expression of PVRIG and PD1 on cells derived from NAT ortumor samples to determine if there is modulated expression in the tumorcompared to healthy tissues (FIG. 15 ). Overall, PVRIG was significantlyincreased on CD4 and CD8 T cells derived from tumor tissue as comparedto matched normal adjacent (FIG. 15A). Within tumor types, PVRIG wasupregulated by at least 2 fold in 3 of 9 colon tumors, 1 or 2endometrium tumors, 4 of 11 kidney tumors, and 4 of 5 lung tumors (FIG.15A). In the same samples, we also evaluated PD-1 expression. Acorrelation analysis between PVRIG fold change (between NAT and tumor)and PD-1 fold change on CD4 and CD8 T cells showed a positive andsignificant correlation in these samples, suggesting that thesemolecules could be co-regulated in similar manner in the tumor.

c. PVRL2 is Expressed on Myeloid and CD45− (Non-Immune) Cells fromMultiple Tumors

In the same samples from which we examined PVRIG expression, we alsoexamined the expression of PVRL2, the ligand for PVRIG. PVRL2 expressionwas detected on 2 major cell subsets, myeloid cells which includemonocytes, mDC, and pDC populations, and CD45- non-immune cells, likelycomposed of tumor epithelium, stromal cells, and endothelial cells (FIG.16 ).

PVRL2 expression on CD45⁻ non-immune cells was detected on cells frombreast, colon/rectum/stomach, endometrial, lung, prostate, and ovariantumors (FIG. 17 ). The highest expression median expression of PVRL2 wasdetected on endometrium and ovarian tumors.

On immune cells, PVRL2 was expressed on myeloid cells from breast,colon/rectum/stomach, endometrium/uterus, head and neck, lung, kidney,prostate, and ovary tissues.

FIG. 18 ). The median expression of PVRL2 on myeloid cells (monocytes,mDCs, pDCs) was comparable across tumor types.

Comparing tumor tissue with normal adjacent tissue, PVRL2 expression wassignificantly increased on tumor CD45− cells or on monocytes from tumortissues (FIG. 19 ). PVRL2 expression on monocytes or CD45− cells wasinduced by at least 2 fold in the tumor compared to normal adjacent in 5of 9 colon tumors, 1 of 2 endometrium tumors, 5 of 11 kidney tumors, 4of 5 lung tumors, and 1 of 1 ovarian tumors. These data supportincreased expression of PVLR2 on tumor cells and on immune cells withinthe tumor. A correlation analysis between of PVRL2 fold change (betweenNAT and tumor) and PD-L1 fold change on CD45− cells and monocytes showeda positive and significant correlation in these samples, suggesting thatthese molecules could be co-regulated in similar manner in the tumor.

d. PVRIG and PVRL2 are Co-Expressed in the Same Tumor

We further assessed which tumor types have high expression of both PVRIGon T cells and PVRL2 on either monocytes and tumor cells. In this sampleset, tumors from endometrium, lung, and kidney tissues displayed highexpression of both PVRIG on T cells and PVRL2 on either monocytes orCD45− cells (FIG. 20 ), suggesting that these tumor types may be moreresponsive to CHA.7.518.1.H4(S241P) treatment.

e. Conclusion

The results from these studies demonstrate that PVRIG is expressed oneffector lymphocytes such as CD8 T cells and NK cells within the tumormicroenvironment. Both PVRIG and PVRL2 were expressed in multiple tumorsamples from breast, colon/rectum/stomach, endometrial, lung, prostate,and ovarian tumors. Expression of PVRIG on T cells was significantlyincreased in tumor tissues as compared to matched normal adjacenttissues. Furthermore, a significant direct correlation was observedbetween PVRIG and PD-1 and PVRIG and TIGIT expression on CD4 and CD8 Tcells from endometrial samples. On a single cell basis, co-expression ofPVRIG with PD1 or with TIGIT was observed on CD8 T cells. The ligand forPVRIG, PVRL2, is expressed on antigen presenting cells (monocytes, mDCs,pDCs) and also on CD45− cells (presumably composed of epithelial,stromal, endothelial cells) from multiple tumor tissues. Induction ofPVRL2 expression was detected on cells derived from tumor as comparedwith normal adjacent tissues. The cellular expression profile of thereceptor and ligand suggest a role for this pathway in regulatingeffector lymphocyte responses for multiple tumor types.

Example 3 Expression of PVRL2 and PD-L1 in Human Cancer and NormalTissues by IHC

The purpose of this study was to examine the expression of PVRL2 andPD-L1 in human healthy and cancer tissue. Two antibodies to PVRL2 wereidentified to stain for PVRL2 in formalin fixed paraffin embedded (FFPE)fixed samples. PD-L1 was assessed using a commercially validatedantibody. Using these antibodies, we examined expression of PVRL2 andPD-L1 in serial tissue sections of a tumor microarray (TMA) composed ofbreast, colon, lung, ovarian, and skin tissues. PVRL2 expression wasobserved to be enhanced in breast, colon, lung, ovarian, and skincancers as compared to healthy tissues. Similar staining was observedbetween the two PVRL2 antibodies, helping to corroborate the resultsobtained. PD-L1 expression was also increased in breast, colon, lung,ovarian, and skin cancers as compared to healthy tissues. Expression ofPVRL2 was observed on tumor epithelium and also on infiltrating immunecells. There was a higher incidence of PVRL2 expression than PD-L1expression in these tumor samples. Individual tumor samples were furthergrouped by PD-L1 negative and positive expression, and PVRL2 expressionanalyzed in these subgroups. All PD-L1 positive tumors also expressedPVRL2, providing a rationale for combination treatment in tumors. InPD-L1 negative tumors, PVRL2 expression was detected in a subset ofthese samples, providing a rationale for targeting the PVRL2 pathway inPD-L1 negative tumors. Taken together, these data demonstrate that PVRL2expression was enhanced in the tumor microenvironment from breast,colon, lung, ovarian, and skin cancer and provide a rationale formonotherapy and combination treatment with agents targeting the PVRL2pathway.

Protocols Antibodies

Anti-PVRL2 (Abcam ab135246, Sigma HPA-012759) and anti-PDL1 (SpringBioSp142) were used in this study. Isotype control antibody (Rabbit IgG)was used as the negative control.

IHC Staining

Breast, colon, lung, ovarian, skin tumor microarrays were obtained from.Each microarray contains healthy tissues from 2-4 donors and tumortissue from 30-40 donors, present in duplicates on the slide. Anti-PVRL2Abcam ab135246 staining was performed at 1:250 dilution with noheat-induced antigen retrieval (HIER). Anti-PVRL2 (Sigma HPA-012759) wasused at 0.1 ug/ml with HIER at pH 9.5. Anti-PD-L1 (SpringBio SP142) wasused at 1 ug/ml with HIER at pH6.2 based on the manufacturer'srecommendation. A matching rabbit IgG isotype control was used at eachof the relevant conditions. Each core was qualitatively scored based on:No staining (score 0), partial positive (score 1), positive (score 2),strong positive (score 3) by 2 individual operators. In cases of a scorediscrepancy between the 2 operators, the sample was reassessed by bothoperators for a final score. The score from the 2 cores derived from thesame tumor were averaged and one score obtained for each tumor. Scoreswere plotted and samples were grouped by pathology data provided by thevendor.

Results and Discussion PVRL2 and PD-L1 Expression are Increased inBreast, Colon, Lung, Ovarian, and Skin Cancers

Two anti-PVRL2 antibodies (ab135246, HPA-012759) were tested for abilityto assess PVRL2 expression in formalin fixed paraffin embedded tissues.Expression of PVRL2 and PD-L1 was assessed serial sections of a tumormicroarray of breast, colon, lung, ovarian and skin cancers. Expressionof PVRL2 and PD-L1 was increased in breast, colon, lung, ovarian, andskin cancers (FIG. 21 ).

PVRL2 is Expressed in PD-L1 Positive and PD-L1 Negative Tumors.

As the expression of PVRL2 and PD-L1 was conducted on serial sections ofthe same TMA, we were able to examine the expression of PVRL2 and PD-L1in each of these tumors from the same portion of the tumor (FIG. 22 ). Asubset of PD-L1 negative tumor samples, in particular lung, ovarian,breast tumors, expressed PVRL2 (as defined by at least partial positive)as detected by both anti-PVRL2 antibodies. These data show that PVRL2can be expressed in PD-L1 negative tumors. In contrast, all PD-L1positive tumors expressed PVRL2 as detected by both PVRL2 antibodies.

PVRL2 is Expressed on the Epithelial Cells and on the Immune Compartmenton the Invasive Front

The spatial expression of immune checkpoints at the invasive front of atumor is important in regulating the anti-tumor response. Knowncheckpoint targets such as PD-1 and PD-L1 have prominent expression atthe invasive front. As these TMAs are generated from punch biopsies anddo not contain the whole tumor, we examined these samples for thepresence of immune infiltrate at the invasive front of the tumor. Weidentified 1 sample where we observed PVRL2 expression in the immuneinfiltrate and on the tumor epithelium (FIG. 23 ). PD-L1 expression wasobserved on the immune infiltrate, further suggesting this could be theinvasive front of a tumor.

Conclusion

These results demonstrate the PVRL2 expression is enhanced in breast,colon, lung, ovarian, skin tumors as compared to healthy tissue from thesame organs. PVRL2 is expressed on both tumor epithelium and oninfiltrating leukocytes. We further demonstrate that all PD-L1 positivetumors express PVRL2, suggesting that agents that target PVRL2 pathwaymay be efficacious in combination with PD-1/PD-L1 inhibitors. Inaddition, PVRL2 expression was detected in PD-L1 negative tumors,suggesting that agents that target PVRL2 pathway may be efficacious inPD-L1 negative tumors.

Example 4 Antitumor Responses of Mono, Dual and Triple CombinationAntibody Treatments in the CT26 Tumor Model Rationale and Objectives

To examine whether antibody blockade of PVRIG, TIGIT and PD-L1 canenhance tumor growth inhibition and survival in a syngeneic mouse tumormodel compared to mono or dual antibody treatments.

Materials and Methods In Vivo Tumor Model

CT26 colon carcinoma cells (ATCC) were cultured in RPMI 1640 with 10%FBS, and 100 ug/mL penicillin/streptomycin. For tumor implantation,5×10⁵ CT26 cells were injected subcutaneously into the right flank offemale, 8-week-old BALB/c mice. Following tumor randomization, theantibodies were administered by intra-peritoneal (i.p.) injection,starting on day 7 post tumor inoculation when tumors reached the volumeof 60-90 mm³, and continued for 3 weeks for a total of 6administrations. Tumor size was measured with electronic caliper every2-3 days and was reported as 0.5×W²×L mm³. . Mice were sacrificed ateither study termination or at the clinical endpoints, including tumorvolume 3250 mm³, tumor ulceration, body weight loss 20%, or moribundappearance.

Antibodies

The chimeric anti-mouse PVRIG antibody (Clone 407, internal production)used in these studies was engineered as a mouse IgG1 (mIgG1) antibody.This antibody was shown to bind to 293HEK cells over-expressing mousePVRIG, and to block the binding of the ligand, mouse PVRL2. Theanti-mouse PD-L1 mIgG1 antibody (Clone YW243.55.S70) was generatedaccording to the description in WO/2010/077634. The anti-mouse TIGITmIgG1 antibody (Clone 11A11) was generated according to the descriptionin WO2016/028656. Synagis IgG1 was used as isotype control and producedinternally. Antibodies were formulated in sterile PBS with low endotoxin(<0.05 EU/mg). The anti-PVRIG antibody was administered at a dose of 10mg/kg, anti-PD-L1 at 5 mg/kg, and anti-TIGIT at 18 mg/kg.

Statistical Analysis

Two-way ANOVA with repeated measures, followed by two--way ANOVA withrepeated measures for selected pairs of groups was determined by JUMPsoftware (Statistical Discoveries™). Analyses of tumor growthmeasurements were performed by comparing tumor volumes measured on thelast day on which all study animals were alive. Statistical differencesin percentage of mice tumor free were determined by a Log RankMantel—Cox test. Values of P<0.05 were considered significant. *p<0.05;p<0.01; p<0.001.

Results

In Vivo Efficacy of Anti-TIGIT and Anti-PVRIG Antibodies in Combinationwith Anti-PD-L1 Antibody

The in vivo efficacy of combinational therapy of mouse PVRIG, TIGIT andPD-L1 blockade was evaluated in mouse syngeneic CT26.WT ectopicsubcutaneous tumor model. Treating tumor bearing mice with theanti-PVRIG antibody in combination with an anti-PD-L1 antibody resultedin tumor growth inhibition (TGI) of 47% compared to isotype control.However, in this study no benefit was observed with the dual combinationof anti-PD-L1 and anti-PVRIG antibodies compared to anti-PD-L1 antibodytreatment alone. The blockade of TIGIT in the triple combination(anti-PVRIG, anti-TIGIT and anti-PD-L1) resulted in a significantimprovement in TGI when compared to other dual combination treatmentswith the anti-TIGIT antibody (anti-PDL-1 + anti-PVRIG+anti-TIGIT, andanti-PDL-1+anti-TIGIT, which corresponded to 29%, 61%, and 55% TGI,respectively) (FIGS. 24A and C). The triple combination resulted inhigher response rates (55% vs 40%) and promoted durable antitumoractivity with a trend for higher survival rate until Day 35 (FIG. 24B).

Example 5 PVRIG Antagonism Enhances T Cell Effector Function and ReducesTumor Growth Abstract

Despite recent advances, the majority of patients do not derive longterm benefit from checkpoint inhibitors. PVRIG is a novel immunesuppressive receptor of the DNAM/TIGIT family and we demonstrate here arole of PVRIG in regulating anti-tumor responses. PVRIG binds to PVRL2and displays significantly enhanced expression on tumor infiltratinglymphocytes as compared to lymphocytes from normal tissues. PVRIGantagonism enhanced human T cell activation and combination of PVRIGwith PD-1 or TIGIT inhibitors further synergistically increasedlymphocyte function. We next addressed the role fo PVRIG in preclinicaltumor models. PVRIG^(−/−) mice displayed significantly increased T cellactivation in vitro and reduced MC38 tumor growth that was mediated byincreased CD8 effector function. Antagonistic anti-PVRIG antibodysignificantly reduced tumor growth in combination with anti-PD-L1 orwhen tested in mice. In summary, we demonstrate that PVRIG-PVRL2 pathwaywas induced in human cancers and that antagonizing PVRIG-PVRL2interactions resulted in increased T cell function and reduced tumorgrowth.

State of Significance

These data demonstrate that PVRIG is a promising target for thetreatment of cancer and provide the rationale for testing a PVRIGinhibitor, CHA.7.518.1.H4(S241P), as a novel cancer immunotherapy agenteither as monotherapy or in combination with TIGIT or PD1 blockade.

Introduction

Increasing evidence demonstrate that endogenous immune responses arecritical in sculpting the initiation, progression, and suppression ofcancer (1) (2). The immune status of patients as well as the content oftumor-infiltrating leukocytes (TILs) within the tumor microenvironment(TME) are key prognostic indicators of not only cancer survival rates,but also how patients respond to therapy (3) (4). T cells are a keycomponent of TILs that can invoke an anti-tumor response, and mostanti-tumor immune responses ultimately rely on the functionality ofeffector lymphocytes cells. An enrichment of CD8 T cells in the TME of apatient's tumor, as well as other factors that bias an immune responsetowards an effective CD8 T cell response such as mutational load and aTh1 polarized TME, are all key prognostic indicators for a favorableanti-tumor immune response (5) (6).

A key observation across many solid tumors is that effector T cells havean activated or ‘exhausted’ phenotype within the TME (7). This indicatesthat although T cells within the TME have initially seen cognateantigen, been activated, and trafficked to the tumor, they aresubsequently not capable of invoking an effective anti-tumor response.Pre-activated or exhausted T cells are defined by increased surfaceexpression of co-inhibitory receptors, such as PD-1 and CTLA-4 (8).Therapeutically targeting these co-inhibitory receptors with antibodiesthat inhibit interactions with their cognate ligands have shownremarkable clinical efficacy in patients with multiple advanced cancers(9). Mechanistically, it has been shown that targeting theseco-inhibitory receptors leads to the expansion of already tumor-reactiveT cells that pre-exist in the TME and to the production of T cell poolswith widened T cell receptor diversity (10) (11) (12). Althoughcheckpoint inhibitors currently in the clinic have revolutionized cancertreatment and demonstrated the power of the immune system in combatingcancer, many patients still relapse and/or do not respond to treatment.Consequently, increased understanding of the immune response in cancerand targeting additional immune-based pathways will lead to additionaltherapeutic treatments.

Among these novel pathways, a group of receptors and ligands within thenectin and nectin-like family are currently under investigation aspotential novel cancer immunotherapies. Receptors within this familyinclude DNAM-1 (CD226), CD96 (TACTILE), TIGIT, and more recently, PVRIG(CD112R) (13) (14) (15). Of these molecules, DNAM is an activatingreceptor within this subfamily, binding to 2 ligands, PVR (CD155) andPVRL2 (CD112), to deliver an activating signal to lymphocytes (16). Tworeceptors in this family have been shown to inhibit human lymphocytefunction, TIGIT, and more recently, PVRIG (17) (18). TIGIT is reportedto have a high affinity interaction with PVR, a much weaker affinity toPVRL2, and has been shown to inhibit both T cell and NK cell responsesby delivering an inhibitory signal into lymphocytes through its ITSMmotif (19) (20). More recently, PVRIG was shown to bind with highaffinity to PVRL2 and to deliver an inhibitory signal through its ITIMmotif (15). In both cases, the affinity of TIGIT to PVR and of PVRIG toPVRL2 is higher than the affinity of DNAM to either PVR or PVRL2,suggesting TIGIT and PVRIG can outcompete PVR and PVRL2 from DNAM,providing an indirect mechanism by which TIGIT and PVRIG can reduce Tcell function. Within this family, PVR is also a ligand for CD96. Thefunction of CD96 has been reported to be inhibitory on mouse lymphocytes(21) but activating on human lymphocytes (22). Based on these data, wepostulate on human lymphocytes that 2 receptors, TIGIT and PVRIG, bindwith high affinity to PVR and PVRL2, respectively, to deliver inhibitorysignals to dampen T cell function.

Although human PVRIG has been shown to inhibit T cells response in onerecent report, the role of PVRIG and PVRL2 in cancer immune surveillanceis not well understood. In particular, the expression profile of thispathway in cancers and the role of PVRIG in regulating CD8 T cellanti-tumor responses has not been reported. Furthermore, functionalcharacterization of the mouse PVRIG gene and the effect of disruptingPVRIG-PVRL2 interaction in vivo in pre-clinical tumor models has notbeen reported. Herein, we elucidated the role of PVRIG in a cancersetting by reporting on PVRIG and PVRL2 expression profile in cancer andthe effect of PVRIG antagonism in tumor cell co-culture assays and inpreclinical tumor models. We demonstrate that PVRIG has a differentiatedexpression profile on T cell subsets compared to TIGIT or CD96 and thatPVRIG and PVRL2 expression were induced in cancer compared to normaladjacent tissues. In multiple human in vitro assay systems, ahigh-affinity PVRIG antagonistic monoclonal antibody(CHA.7.518.1.H4(S241P)) enhanced T cell function, in particular whencombined with anti-TIGIT or anti-PD1 antibody. In addition, we reportthe novel characterization of mouse PVRIG using antagonistic antibodiesor PVRIG deficient mice and demonstrate that inhibition of PVRIG-PVRL2interaction reduced tumor growth, with most potent effects incombination with PD-1 inhibition or TIGIT genetic deficiency.Collectively, this data shows that PVRIG is a critical inhibitoryreceptor in regulating T cell anti-tumor responses and support thedevelopment of CHA.7.518.1.H4(S241P), for clinical testing in cancerpatients.

Materials and Methods Human Peripheral Blood and Tumor ExpressionStudies

Healthy donor human PBMCs were obtained from Stanford University inaccordance with the Declaration of Helsinki. Human tissues were providedby the Cooperative Human Tissue Network, a National Cancer Institutesupported resource. Human cancer tissue and matched normal adjacenttissues Were dissociated into single cells as per manufacturer'sprotocol (Miltenyi Biotec). Dissociated cells were analyzed by flowcytometry for expression of various targets on different cell subsets.For each target expression on an individual cell subset, a foldexpression value was calculated by taking the MFI value of targetdivided by the MFI value of the isotype control. Other investigators mayhave received samples from these same tissue specimens. The tumor typewas determined based on reviewing the pathology report for each sample.For IHC studies, anti-PVRL2 antibody (HPA-012759, Sigma) and PD-L1(Sp142, SpringBio) were used to stain tumor micro-arrays (Biochaininstitute) using conditions as described in the supplemental methods.Scoring was performed by 2 independent reviewers on duplicate cores fromthe same tumor.

PVRIG Antibody Generation and Characterization

Anti-human PVRIG and anti-mouse PVRIG antibodies were generated asdetailed in the supplemental methods. Briefly, antibody bindingspecificity and affinity Were assessed by selective binding to PVRIGengineered cells with no detectable binding to cells that do no expressthe gene. Antagonistic activity of these anti-PVRIG antibodies wasdetermined using ELISA and FACS based assays in which the interaction ofPVRIG with PVRL2 was disrupted. For characterization in cell basedassays, antibodies were tested in several T cell-target cell co-cultureassay systems consisting of target cells that express PVRL2 in culturewith PBMC or tumor-derived T cells. gp100 specific T cells lines wereexpanded from melanoma tumors as previously described (23). CMVpp65reactive T cells were expanded from healthy donor PBMCs (CTL immunospot)with CMVpp65 (495-503), 11,-2, and 1L-7 for 10 days. For combinationstudies, antibodies to PD-1, TIGIT, and PVRIG were used at 10 μg/ml.Cytokine concentrations in conditioned media was determined usingCytometric Bead Array (CBA) and FACS staining was performed as describedin the supplemental methods.

Characterization of Mouse PVRIG Expression and Function

Binding interactions of mouse PVRIG with mPVRL2 and mPVR were assessedby SPR and ELISA using recombinant PVRIG, PVRL2, and PVR proteins and byFACS using ectopically engineered PVRIG and P1/RL2 overexpressing celllines or PVR or PVRL2 siRNA transfected cell lines. PVRIG and TIGITdeficient mice were generated as described in the supplemental methods.Expression analysis was performed to examine expression of PVRIG inspleen, lymph node, and tumor in various cell subsets. Cell functionalassays demonstrating a T cell modulatory activity for mouse PVRIG wereestablished using WT and PVRIG T cells and PVRL2 Fc or PVRL2 ectopicallyexpressed target cells as detailed in the supplemental materials andmethods. CT26, MC38, and B16/Db-hmgp100 tumor models were performed asdescribed in the supplemental methods. All studies were approved by theInstitutional Animal Care and Use committee at the Tel-Aviv University(Tel-aviv, Israel) or Johns Hopkins University (Baltimore, USA).

Results PVRIG Expression is Highest on Effector T Cells of PeripheralBlood and Tumors

The Ig superfamily (IgSF) consists of hundreds of proteins but only afew of them are T cell inhibitory receptors. Proteins of the IgSF tendto evolve quickly (24) and therefore sequence similarity among theseproteins is generally low and is not optimal for identifying novelimmune receptors. To identify novel immune checkpoints, we developedbioinformatic algorithms based on shared genomic and proteomiccharacteristics among known immune checkpoints, such as gene structure,protein domains, predicted cellular localization and expression pattern.Using these algorithms, we identified PVRIG as a novel immune receptor.A report has recently also demonstrated that human PVRIG (CD112R) bindsto PVRL2 and inhibits T cell function (15). However, the relevance ofthis pathway in regulating tumor immune surveillance has not beenreported. Here, we have elucidated the expression and function of PVRIGand PVRL2 in human cancers and preclinical tumor models. In peripheralblood from healthy donors, PVRIG was expressed exclusively onlymphocytes, with highest expression on CD8 T cells and NK cells (FIG.27A). Further subset analysis of T cells showed highest PVRIG expressionon CD8 or CD4 memory/effector T cell subsets in comparison with Tregsubset (FIG. 27B, FIG. 34A). The predominantly memory T-cell expressionpattern differentiates PVRIG from other receptors in the family (TIGIT,CD96) which tend to have equal or higher expression on Tregs compared tomemory/effector T cells. We further compared the expression kinetics ofPVRIG and TIGIT post T cell activation in 2 assay systems (CMV recallresponse FIG. 27C, DC-MLR FIG. 27D, FIG. 34B) and show that PVRIG hasdelayed kinetics of induction and more sustained expression at the latetimepoint as compared to TIGIT. The preferential expression of PVRIG onmemory/effector cells as compared to TIGIT suggests a unique role forPVRIG in regulating T cell responses.

The delayed and sustained induction of PVRIG expression on T cells afteractivation suggested that it could be expressed in the tumormicroenvironment. Next, we analyzed the expression of PVRIG onleukocytes from dissociated human tumors directly ex vivo by FACS.Expression of PVRIG was detected on CD8 T cells, CD4 T cells, and NKcells from multiple tumor types (FIG. 27E-G, Supplemental FIG. 27C).PVRIG was co-expressed with PD-1 and TIGIT on CD4 and CD8 T cells (FIG.27F) On average, higher expression was detected on CD4⁺ and CD8+ TILsfrom breast, endometrial, head and neck, lung, kidney, and ovariantumors as compared to bladder, colorectal, and prostate. In tumorsamples in which PVRIG expression was low/not present ex vivo,activation with anti-CD3 and anti-CD28 enhanced the expression of PVRIG,suggesting that TIL expression of PVRIG can be further induced uponre-activation (FIG. 34D). For colon, lung, kidney, endometrial, andovarian tumors, we were able to obtain normal adjacent tissue from thesame patient and perform a comparison of PVRIG expression on lymphocytesisolated from the tumor vs normal tissue. TILS showed a significantinduction of PVRIG on CD4 and CD8 T cells as compared to cells isolatedfrom matching normal adjacent tissues (NAT) (FIG. 34E). As with PBMCs,we further compared PVRIG, TIGIT, and PD1 expression on Tregs vs CD8 Tcells from lung, endometrial, and kidney tumors. On TILS, TIGITexpression was higher on Tregs compared to CD8 T cells whereas for PVRIGand PD1, similar or higher expression was observed on CD8 T cellscompared to Tregs (FIG. 27E). Next, we examined the co-regulation ofPVRIG, TIGIT, and PD-1 on T cell populations by correlation analysis ofeither the magnitude of expression on TILS ex vivo or the magnitude ofthe fold change in expression between tumor and NAT. In both analyses,CD4 and CD8 T cells displayed a positive and significant correlationbetween PVRIG and PD1 or TIGIT on (FIG. 34F). Taken together, these datademonstrate that PVRIG is expressed on T cells and NK cells frommultiple human cancers, placing PVRIG as a novel inhibitory receptortarget that may be critical in regulating T cell function in the tumor.

PVRL2 Expression is Enhanced in Tumors Tissue Compared to NormalAdjacent Tissue

As PD-L1 expression has been demonstrated to help predict responses toPD-1 inhibitors, we examined whether the expression of PVRL2 wasconcomitant with expression of its cognate receptor, PVRIG, in humancancer tissues. Using a PVRL2 antibody that we validated for use in IHC(FIG. 35A), we stained tumor microarrays (TMA) composed of lung, colon,skin, breast, ovarian/endometrial, and kidney cancer tissues. With theexception of kidney, PVRL2 expression was not present or minimallyexpressed in the majority of normal tissues from these organs. In tumortissues, PVRL2 was induced in a substantial number of lung, colon, skin,breast, and ovarian/endometrial cancer samples (FIG. 28A). PVRL2expression was detected on tumor cells and immune cells at the invasivefront (FIG. 28B). To determine the specific immune cell subsetsexpressing PVRL2, we performed flow cytometry on freshly dissociatedtumors. Consistent with the IHC expression profile, expression of PVRL2was detected on CD45⁺ immune cells, particularly myeloid cells (e.g.CD14⁺ tumor associated macrophages (TAMs) and myeloid DCs) and onCD45⁻non-immune cells from multiple tumor types (FIG. 28C, D). Noexpression of PVRL2 was detected on lymphocytes (data not shown).Comparison of PVRL2 expression on CD45⁻cells and TAMs isolated fromcolon, lung, kidney, endometrial, and ovarian tumors showed asignificant induction of PVRL2 on cells isolated from the tumor ascompared to cells isolated from matching normal adjacent tissues (NAT)of the same donor (FIG. 36D). To assess which tumors expressed bothPVRIG and PVRL2, we examined expression of PVRIG on lymphocytes comparedwith PVRL2 on myeloid cells and on CD45⁻cells from multiple tumor types.Of the cancer types examined, endometrial, lung, and kidney cancers hadthe highest prevalence of PVRIG^(hi) lymphocytes and PVRL2^(hi) TAMs orCD45⁻non-immune cells (FIG. 28E, FIG. 37 ). These data demonstrate thatthe PVRIG-PVRL2 pathway may be particularly important in modulating theanti-tumor response by regulating the T cell—TAM interaction and the Tcell-tumor cell interaction in endometrial, lung, and kidney cancers.

Compared to PD-L1, PVRL2 Expression is Differentially Regulated andPresent in PD-L1⁻Tumors

As PVRIG and PD-1 can be co-expressed on tumor-infiltrating lymphocytes(TILs), we also examined the co-expression of PVRL2 and PD-L1 on thesame tumor by staining serial sections of the same TMA. AllPD-L1-positive tumors also expressed PVRL2, indicating some overlap inthe regulation of these 2 pathways and providing a rationale to combinea PVRIG inhibitor with PD-1/PD-L1 inhibitors (FIG. 29A). InPD-L1negative tumors, PVRL2 was detected in a majority of these tumorsacross various cancer types (FIG. 29A). This suggested that PVRL2expression was more prevalent than PD-L1 in some tumors and thattargeting this pathway may be particularly effective in PD-L1-negativetumors. As PD-L1 has been reported to be induced in the tumor by IFN-γas part of the adaptive resistance model (25), we further assessed theregulation of PVR, PVRL2 and PD-L1 expression by various inflammatorystimuli on bone marrow derived dendritic cells and on tumor epithelialcell lines (FIG. 29D). Treatment of immature BM-DCs withpro-inflammatory signals generally lead to an increase in PVR, PVRL2,and PD-L1 expression, demonstrating that PVR, PVRL2, and PD-L1expression are both increased upon DC maturation. In contrast, treatmentof epithelial cells with IFN-γ increased expression of PD-L1 but had noeffect on the high baseline expression of PVRL2 (FIG. 29E). It has beenreported that PVRL2 by genomic stress, DNA damage, and tumor suppressorgenes (26) (27), further supporting a differential regulation of PVRL2expression in comparison with PD-L1. In summary, these data indicatethat PD-L1 and PVRL2 can be co-regulated on antigen presenting cellssuch as DCs but can be differentially regulated by IFN-γ on epithelialcells. The presence of PVRL2 in PD-L1-negative tumors suggests thattargeting this pathway may be of potential benefit in patients that arenon-responsive to PD-1 inhibitors.

CHA.7.518.1.H4(S241P) is a High Affinity Humanized Monoclonal Antibodyto PVRIG that Disrupts the Interaction of PVRIG to PVRL2

To examine the functional consequences of antagonizing human PVRIG-PVRL2interactions, we generated a high affinity, antagonistic anti-PVRIGantibody, CHA.7.518.1.H4(S241P), which blocks the interaction of PVRIGand PVRL2. This antibody selectively bound HEK293 cells ectopicallyexpressing human PVRIG or cynomolgus macaque PVRIG and also bound Jurkatcells that endogenously express PVRIG with sub-nanomolar affinity (FIG.30A). In biochemical assays, CHA.7.518.1.H4(S241P) blocked theinteraction of PVRIG Fc with PVRL2⁺ HEK293 cells (FIG. 30B) and alsoblocked PVRL2 Fc binding to PVRIG HEK293 cells (FIG. 30C). Using thisantibody, we observed a functional effect of an antagonistic anti-PVRIGin several T cell assays. Artificial antigen-presenting cells (aAPC)ectopically expressing a cell surface anti-CD3 antibody and human PVRL2were generated and co-cultured with primary human CD4 T cells, either inthe presence of anti-PVRIG (CHA.7.518.1.H4(S241P)) or isotype control.PVRIG expression was induced on proliferating CD4 T cells uponco-culture with the CHO anti-CD3 aAPC (FIG. 38A). Antagonism of PVRIGwith CHA.7.518.1.H4(S241P) enhanced proliferation of CD4 T cells frommultiple donors (FIG. 30D). We also tested the effect of anti-PVRIG on 2human gp100 reactive CD8 T cell lines that were derived from melanomatumors. These T cell lines were individually co-cultured with aAPCsexpressing HLA-A2 and PVRL2 (FIG. 38B) in the presence of isotypecontrol IgG or anti-PVRIG antibodies. As observed in both lines,anti-PVRIG increased IFN-γ and TNF-α production by ˜20-50%. In a doseresponse assessment, CHA.7.518.1.H4(S241P) displayed single digitnano-molar EC50 values in multiple assays (FIG. 38C, D). These datacollectively demonstrate that antagonizing PVRIG-PVRL2 interactions withCHA.7.518.1.H4(S241P) resulted in increased T cell activation.

CHA.7.518.1.H4(S241P) in Combination with TIGIT or PD-1 InhibitorsResulted in Synergistic Enhancement of T Cell Function.

Combination of PVRIG and TIGIT blockade synergistically increased CD4 Tcell function in a T cell-dendritic cell co-culture assay (15),suggesting a role for this pathway in regulating T cell-APCinteractions. The effects of PVRIG and TIGIT blockade on CD8 T cells ina tumor cell co-culture setting has not been reported. As our tumorexpression profiling demonstrated expression of PVRL2 on CD45⁻ immunecells, we further explored the effect of targeting this pathway in Tcell—tumor cell co-cultures using 2 T cell assay systems. We firstperformed a co-culture of 2 gp100 tumor antigen specific CD8 T celllines with a melanoma cell line, MEL624, in the presence of anti-PVRIG,anti-TIGIT, or isotype control antibodies, either individually or incombination. MEL624 cells express both PVR and PVLR2 and both TIL-209and TIL-463 expressed PVRIG, TIGIT, and PD-1 (FIG. 30F). On TIL-209, weobserved that anti-PVRIG or anti-TIGIT alone did not increase IFN-y andthat the combination of anti-PVRIG and anti-TIGIT synergisticallyincreased IFN-y production (FIG. 30G). On TIL-463, we observed thatanti-PVRIG or anti-TIGIT modestly increased IFN-γ production, and thatcombination of anti-PVRIG and anti-TIGIT additively increased IFN-γ(FIG. 30G). In an additional assay system, we utilized CMVpp65-reactiveCD8 T cells as a model system to study human T cell responses. HLA-A2+CMVpp65 CD8 T cells were expanded in the presence of CMVpp65 (495-503)and expression of PVRIG, TIGIT, and PD-1 was observed on day 10 (FIG.30F). PVRIG was expressed on CMVpp65 specific CD8 T cells at similarmagnitude to what was observed in human cancer samples (FIG. 27 ). Astarget cells, we identified a PD-L1^(hi) (Panc05.04) and a PD-L1^(lo)(Colo205) HLA-A2⁺ cancer cell line that both expressed similar amountsof PVR and PVRL2 (FIG. 30F). We next performed a co-culture of theCMVpp65 reactive T cells with HLA-A2⁺ tumor cell lines pulsed with pp65(495-503) peptide in the presence of blocking antibodies to PVRIG,TIGIT, and/or PD-1. We observed that anti-PVRIG Ab increased IFN-≢5 by˜50% in the co-culture with Panc05.04 cells and minimally in theco-culture with Colo205 (FIG. 30H). Combination of anti-TIGIT withanti-PVRIG Ab synergistically increased IFN-γ production on both targetcell lines, resulting in a greater increase in IFN-γ compared to PD-1antibody alone (FIG. 30H). Combination of anti-PVRIG and anti-PD-1 alsoled to synergistic increases in IFN-γ production as compared toindividual antibody (FIG. 30I). Taken together, these data suggest apotent synergy of combining PVRIG and TIGIT or PVRIG and PD1 blockade inincreasing activation of human CD8 T cells upon interaction with tumorcells.

PVRIG Deficiency Resulted in Increased T Cell Proliferation and ReducedTumor Growth

Although the sequence for mouse PVRIG and its interaction with mousePVRL2 has been reported, the expression profile and immune modulatoryactivity of mouse PVRIG is not well understood. We first analyzed mPVRIGRNA expression and transcript in NK, NKT and T cells (FIG. 31 .A).Activated mouse CD8 T cells had elevated PVRIG transcripts with delayedinduction kinetics compared to TIGIT (FIG. 31B). We confirmed that thatrecombinant mPVRIG bound to mPVRL2 protein by surface plasmon resonance(SPR) and ELISA performed in several assay orientations (FIG. 39A-D), Wealso observed an interaction between mPVRIG and mPVR, although theaffinity was approximately 10× less than the interaction with mPVRL2(FIG. 39E). To determine whether PVR or PVRL2 is the dominant ligand formPVRIG, we tested the binding of mouse PVRIG Fc to B16F10 cells whichexpress PVR and PVRL2 (data not shown). PVRIG Fc showed a dose dependentbinding to B16F10 cells that was completely abolished upon PVRL2 siRNAknockdown in B16F10 cells (FIG. 39F). In comparison, the binding ofPVRIG Fc fusion protein was slightly, but consistently, reducedfollowing PVR knockdown (FIG. 39E) suggesting that a very weakinteraction occurs between mPVRIG and mPVR. Taken together, theseresults demonstrate that in mice, PVRL2 is the primary ligand for PVRIG,as is the case in human. To delineate the role of PVRIG in immuneresponses, we generated PVRIG deficient (^(−/−)) mice (FIG. 40 ).PVRIG^(−/−) mice were born at the expected Mendelian ratios, displayedno overt phenotype up to 10 months of age, and at 8 weeks of age hadsimilar leukocyte cellularity (peripheral and lymphoid tissue) whencompared to wild type mice (FIG. 41 ). Wild-type (WT) CD8 T cells and NKcells express PVRIG and no expression of PVRIG was detected onPVRIG^(−/−) cells (FIG. 31C). To examine the role of PVRIG in regulatingmouse T cell responses, we examined the proliferation of WT andPVRIG^(−/−) T cells in 2 assay systems. WT or PVRIG^(−/−) T cells wereactivated with immobilized anti-CD3 in the presence of soluble PVRL2 Fcor control Fc protein. Soluble PVRL2 Fc significantly inhibited WT CD4⁺T cell proliferation but not PVRIG^(−/−) CD4⁺ T cell proliferation (FIG.31D), suggesting that PVRIG^(−/−) cells lack an inhibitory signal. Toevaluate the role of mouse PVRIG in CD8⁺ T cell interaction with tumorcells, PVRIG^(−/−) mice were bred to panel. TCR transgenic mice, whichexpress a transgenic TCR specific to gp100₂₅₋₃₃ (28). ActivatedPVRIG^(−/−) or WT Pmel CD8+ T cells were co-cultured with B16-Db/gp100melanoma tumor cells that endogenously express PVRL2 (data not shown)and activation and effector function evaluated. PVRIG^(−/−) pmel CD8⁺ Tcells showed enhanced degranulation and production of effector cytokines(IFN-γ and TNF-α) compared to WT cells (FIG. 31E). These data indicatethat mouse PVRIG inhibits activation and effector function oftumor-specific T cells upon co-culture with PVRL2⁺ tumor target cells.

We next studied the effects of PVRIG deficiency on tumor growth in theMC38 syngeneic model. PVRIG^(−/−) mice displayed significantly reducedtumor growth compared to WT mice (FIG. 32A-B), Moreover, PVRIG^(−/−)mice exhibited additional anti-tumor responses following PD-L1 blockadebeginning day 14, reflected in significant (p=0.052) tumor growthinhibition compared to anti-PD-L1-treated WT mice or PVRIG^(−/−) micetreated with isotype control FIG. 32C,D). Consistent with the reducedtumor growth, anti-PD-L1-treated PVRIG^(−/−) mice displayed asignificant increase in IFN-γ⁺TNF-α⁺ effector CD8 T cells upon ex vivostimulation, compared to anti-PD-L1-treated wild-type mice as well asisotype-treated PVRIG^(−/−) mice (FIG. 32E). Further, anti-PD-L1-treatedPVRIG^(−/−) mice also had elevated numbers of effectorcytokine-producing CD8⁺ tumor-infiltrating lymphocytes (TILs), whencompared to anti-PD-L1-treated wild-type mice as well as isotype-treatedPVRIG^(−/−) mice (FIG. 32F). Transcriptomic profiling of CD45⁺ immunecells from tumors harvested midway through the experiment (day 18; micereceived 2 doses of anti-PD-L1 or isotype control) showed that the genesignatures for TIL numbers and cytotoxic TILs were significantlyenhanced in anti-PD-L1-treated PVRIG deficient mice relative to theirwild-type counterparts (FIG. 32G-H). Significant changes in T cellmediated genes (GRZB, IFN-γ) was observed in the PVRIG^(−/−)+ anti-PD-L1group as compared to the other groups (Supplemental FIG. 42 ). Takentogether, these data demonstrate that PVRIG deficiency, particularlywhen combined with PD-L1 blockade, resulted in increased T cellactivation and reduced tumor growth in vivo.

Anti-mPVRIG Antibody Inhibited Tumor Growth in Combination with PD-1Antibody or TIGIT Deficiency

After demonstrating that genetic deficiency of PVRIG resulted in reducedtumor growth, we next aimed to demonstrate that antibody-mediatedinhibition of PVRIG-PVRL2 interaction could improve anti-tumor immunity,in particular in combination with PD1 or TIGIT inhibitors as our humanin vitro data has demonstrated. To assess this, we generated a highaffinity, antagonistic anti-mPVRIG antibody. Affinity assessments ofanti-mPVRIG mAb determined by FACS showed sub-nano-molar Kd (0.33 nM onHEK293 mPVRIG, 0.39 nM on D10.G4.1 cells), similar toCHA.7.518.1.H4(S241P) (FIG. 39F-G). The specificity of this antibody wasfurther confirmed as the majority of binding to D10.G4.1 cells wasabrogated upon mPVRIG knockdown (FIG. 39H). Anti-mPVRIG was tested fordisrupting mPVRIG-mPVRL2 interaction by inhibiting the binding of mPVRIGFc to B16F10 and the binding of mPVRL2 Fc to mPVRIG-overexpressingHEK293 cells (FIG. 33A). Complete blocking of PVRIG-PVRL2 interaction byanti-mPVRIG antibody was observed in both assay formats (FIG. 33A, FIG.39I), demonstrating an antagonistic anti-mPVRIG antibody. Next, wetested the in vivo efficacy of mPVRIG blockade in a syngeneic CT26subcutaneous colon tumor model. PVRIG expression was elevated on NK andT cells in the tumor microenvironment, compared to corresponding splenicor draining lymph node subsets (FIG. 33B). Treating tumor bearing micewith anti-mPVRIG blocking mAb as monotherapy failed to reduce tumorgrowth (data not shown). However, combination of anti-PVRIG andanti-PD-L1 mAbs effectively delayed CT26 tumor growth (FIG. 33C) andincreased significantly the survival of treated mice with 40% rate ofcomplete responders (FIG. 33D). Consistent with our human T cell assaydata, these data demonstrate that combination of PD-1 and PVRIGinhibitors can reduce tumor growth.

We also tested the effect of ablating both PVRIG and TIGIT signaling inregulating anti-tumor responses. For these studies, we tested theefficacy of anti-mPVRIG antibody in either WT or TIGIT^(−/−) miceinoculated with B16F10/Db-hmgp100 melanoma cells. Treatment of tumorbearing WT mice with anti-mPVRIG blocking mAb had minor effect comparedto isotype treatment (17% TGI at day 11 and 8% TGI at endpoint, day 18).The effect of TIGIT deletion on tumor growth was minor as well, comparedto WT control group (17% TGI at day 11 and 13% TGI at endpoint).However, when TIGIT deletion was combined with anti-PVRIG mAb treatment,a significant tumor growth inhibition was observed (63% at day 11 and49% TGI at endpoint (FIG. 33E, F). In accordance to tumor growthinhibition, TIGIT^(−/−) mice treated with anti-PVRIG mAb 407 exhibitedincreased survival compared to NT control group, however, statisticalsignificance was not achieved in this aggressive rapidly growing tumormodel (data not shown). Taken together, these data demonstratesynergistic activity of PVRIG inhibitors with PD1 or TIGIT inhibitorsand are in accordance with our human functional data providing therationale for clinical testing of CHA.7.518.1.H4(S241P) with PD1 orTIGIT inhibitors.

Discussion

Although antibodies targeting immune T cell checkpoints such as CTLA4and PD-1 have increased cancer patient survival, the majority of cancerpatients still do not display clinical benefit. One possible reason forthis is the presence of additional T cell regulators that inhibit T cellanti-tumor immunity. Here, we elucidated the role of PVRIG in regulatingeffector T cell function and demonstrate that PVRIG antagonism increasesT cell anti-tumor responses and reduces tumor growth.

PVRIG is a novel member of the nectin and nectin like family, placing itamong several known immunoregulatory receptors in the family.Understanding the interplay of the receptors within this family iscrucial to understanding the relevance and mechanism of action of PVRIG.Of these receptors, DNAM, TIGIT, and CD96 are most closely related toPVRIG in terms of sharing the same ligands, PVR and PVRL2. DNAM binds toboth PVR and PVRL2 and delivers a costimulatory signal to lymphocytes.TIGIT is reported to bind to PVR and weakly to PVRL2. We were unable todetect an interaction between TIGIT and PVRL2 using ELISA or SPR (datanot shown), suggesting that PVR is the dominant ligand for TIGIT. Usingsimilar methods, we and a recent report detected a high affinityinteraction between PVRL2 and PVRIG, suggesting that PVRIG is thedominant inhibitory receptor to PVRL2. These data suggest that TIGIT andPVRIG comprise dual signaling nodes in this axis and that blocking bothis needed for maximal increase of T cell activation within this family.In addition to interacting with different ligands, we observed thatPVRIG has the highest expression on effector or memory T cells, similarto PD-1 whereas TIGIT has the highest expression on regulatory T cells.Furthermore, we observed that PVRIG displayed late induction after Tcell activation as compared to TIGIT. These data suggest that PVRIG hasa unique role within this family, interacting with high affinity toPVRL2 and having a differentiated expression on memory cells and a lateinduction profile to TIGIT.

We also report here on the novel role of PVRIG in regulating anti-tumorT cell responses using PVRIG deficient mice and antagonistic anti-PVRIGantibodies. We demonstrate that mouse PVRIG was expressed on T cells andNK cells, induced upon lymphocyte activation, and is highest in the TMEas compared to the periphery. Furthermore, we show that PVRIG deficiencyled to increased T cell function in-vitro and reduced tumor growthin-vivo. An antagonistic antibody to PVRIG reduced tumor growth whencombined with anti-PD-L1 or genetic deficiency of TIGIT, demonstrating anecessary role of PVRIG in regulating T cell responses. These novel dataprovide in vivo proof of concept using preclinical tumor models thattargeting PVRIG in combination with PD1 or TIGIT antagonism is apotential novel therapy for the treatment of cancers.

We report here on a high affinity anti-human PVRIG antibody thatdisrupts the interaction of PVRIG and PVRL2 which we are pursuing fortesting in clinical trials. To determine potential cancer indicationsthat could inform on patient selection in clinical trials, we examinedthe expression profile of this axis in human cancers by FACS and IHC.For PVRIG, we observed that the mean expression of PVRIG on CD4 and CD8T cells by FACS highest in endometrial, lung, kidney, and ovariancancers, although this difference did not achieve statistical differencewith other cancer types as determined by ANOVA with a Tukey's multiplecomparison test with the current number of samples. As PVRIG is inducedupon T cell activation and given that the majority of tumor infiltratingT cells are antigen experienced, it is perhaps not surprising that themedian PVRIG expression was similar across tumor samples and cancertypes. We observed that PVRIG expression was correlated with PD-1 andTIGIT expression, suggesting that the interplay of these 3 inhibitoryreceptors will be important in regulating the anti-tumor response. Inthis report, we observed a synergistic increase in T cell function whenPVRIG antibodies were combined with TIGIT antibodies in a CD8 T celltumor cell co-culture, better than PD-1 in combination with PVRIG orTIGIT inhibitors. These data, along with a previous study demonstratinga role for PVRIG and TIGIT in regulating DC-T cell interactions, showthat this pathway could be involved in regulating T cell-APC and Tcell-tumor cell interactions, and provide multiple mechanisms by whichtargeting PVRIG could increase the anti-tumor immune response.

As expression of PD-L1 has been correlated with clinical response toPD-1 inhibitors, we also analyzed PVRL2 expression in tumors by FACS andIHC to assess whether certain cancer types have higher expression.Assessing dissociated tumor cells, we observed that mean PVRL2expression on macrophages from endometrial, head & neck, kidney, lung,and ovarian samples were higher when compared to other tumor types. MeanPVRL2 expression on CD45⁻non immune cells was higher on breast,colorectal, endometrial, lung, ovarian, and prostate cancers compared toother cancers. Based on the PVRIG and PVRL2 expression, we determinedthat endometrial, head & neck, lung, kidney, and ovarian cancers have agreater incidence of tumors with high PVRIG and PVRL2 expression andthat these are potential cancers that could response to inhibitors ofthis pathway.

We did observe that PVRL2 expression can be modulated on antigenproducing cells in vitro by inflammatory mediators whereas PVRL2expression on cancer cells was not altered. These data suggest thatPVRL2 expression on antigen presenting cells can be regulated byinflammation and could be an indicator of an inflamed tumor. Indeed, wedid observe that all PD-L1+ tumors also express PVRL2, both on the tumorcells and in the immune compartment. Expression of PVRL2 on myeloidcells could help predict responses to PVRIG inhibitors in a combinationsetting with PD-1 or TIGIT to further enhance the anti-tumor effect.Interestingly, a portion of PD-L1 negative tumors also expressed PVRL2,primarily on the tumor cells and not on the immune cells. PVR and PVRL2expression on epithelial cells is reported to be induced intumorigenesis by xyz and also in response to stress and DNA damage.These data are consistent with our in vitro finding that the regulationof PVRL2 expression on tumor cells is not dependent on IFN-γ. As PD-L1is induced in an adaptive resistance setting in response to IFN-γ and isassociated with an inflammatory response, the expression of PVRL2 in theabsence of PD-L1 suggests that PVRL2 expression is more prevalent thanPD-L1 and that PVRL2 is expressed in non-inflamed tumors. Based on theabove, it is possible that the presence of PVR and PVRL2 contribute tosuppressing immune responses independently of PD-L1 and that inhibitorsof PVRIG and TIGIT could be of particular importance in patients thatare PD-L1 negative or non-responders/progressors to PD-1 inhibitors.

In summary, this report provides several novel insights into PVRIGbiology, including characterizing the expression of this axis in humancancers, demonstrating a prominent role for PVRIG/TIGIT in regulatingthe CD8-tumor cell interaction, and showing that PVRIG antagonism incombination with PD-1 inhibition or TIGIT deficiency lead to asynergistic reduction in tumor growth. These data extend our currentunderstanding of PVRIG biology and provide rationale for clinicaltesting of CHA.7.518.1.H4(S241P), a high affinity anti-PVRIG antibody,in patients with cancer.

REFERENCES

-   1. Hanahan D, Weinberg R A. The hallmarks of cancer. Cell 2000;    100(457-70.-   2. Hanahan D, Weinberg R A. Hallmarks of cancer: the next    generation. Cell 2011; 144(5):646-74 doi 10.1016/j.ce11.2011.02.013.-   3. Galon J, Mlecnik B, Bindea G, Angell H K, Berger A, Lagorce C, et    al. Towards the introduction of the ‘Immunoscore’ in the    classification of malignant tumours. J Pathol 2014; 232(2):199-209    doi 10.1002/path.4287.-   4. Zitvogel L, Galluzzi L, Smyth M J, Kroemer G. Mechanism of action    of conventional and targeted anticancer therapies: reinstating    immunosurveillance. Immunity 2013; 39(474-88 doi    10.1016/j.immuni.2013.06.014.-   5. Danilova L, Wang H, Sunshine J, Kaunitz G J, Cottrell T R, Xu H,    et al. Association of PD-1/PD-L axis expression with cytolytic    activity, mutational load, and prognosis in melanoma and other solid    tumors. Proc Natl Acad Sci USA 2016; 113(48):E7769-E77 doi    10.1073/pnas.1607836113.-   6. Topalian S L, Taube J M, Anders R A, Pardoll D M.    Mechanism-driven biomarkers to guide immune checkpoint blockade in    cancer therapy. Nat Rev Cancer 2016; 16(5):275-87 doi    10.1038/nrc.2016.36.-   7. Zarour H M. Reversing T-cell Dysfunction and Exhaustion in    Cancer. Clin Cancer Res 2016; 22(8):1856-64 doi    10.1158/1078-0432.CCR-15-1849.-   8. Pardoll D M. The blockade of immune checkpoints in cancer    immunotherapy. Nat Rev Cancer 2012; 12(4):252-64 doi    10.1038/nrc3239.-   9. Sharma P, Allison J P. Immune checkpoint targeting in cancer    therapy: toward combination strategies with curative potential. Cell    2015; 161(2):205-14 doi 10.10164 cell.2015.03.030.-   10. Cha E, Klinger M, Hou Y, Cummings C, Ribas A, Faham M, et al.    Improved survival with T cell clonotype stability after anti-CTLA-4    treatment in cancer patients. Sci Transl Med 2014; 6(238):238ra70    doi 10.1126/scitranslmed.3008211.-   11. Robert L, Tsoi J, Wang X, Emerson R, Homet B, Chodon T, et al.    CTLA4 blockade broadens the peripheral T-cell receptor repertoire.    Clin Cancer Res 2014; 20(9):2424-32 doi    10.1158/1078-0432.CCR-13-2648.-   12. Tumeh P C, Harview C L, Yearley J H, Shintaku I P, Taylor E J,    Robert L, et al. PD-1 blockade induces responses by inhibiting    adaptive immune resistance. Nature 2014; 515(7528):568-71 doi    10.1038/nature13954.-   13. Chan C J, Andrews D M, Smyth M J. Receptors that interact with    nectin and nectin-like proteins in the immunosurveillance and    immunotherapy of cancer. Curr Opin Immunol 2012; 24(2):246-51 doi    10.1016/j.coi.2012.01.009.-   14. Martinet L, Smyth M J. Balancing natural killer cell activation    through paired receptors. Nat Rev Immunol 2015; 15(4):243-54 doi    10.1038/nri3799.-   15. Zhu Y, Paniccia A, Schulick A C, Chen W, Koenig M R, Byers J T,    et al. Identification of CD112R as a novel checkpoint for human T    cells. J Exp Med 2016; 213(2):167-76 doi 10.1084/jem.20150785.-   16. Bottino C, Castriconi R, Pende D, Rivera P, Nanni M, Carnemolla    B, et al. Identification of PVR (CD155) and Nectin-2 (CD112) as cell    surface ligands for the human DNAM-1 (CD226) activating molecule. J    Exp Med 2003; 198(4):557-67 doi 10.1084/jem.20030788.-   17. Yu X, Harden K, Gonzalez L C, Francesco M, Chiang E, Irving B,    et al. The surface protein TIGIT suppresses T cell activation by    promoting the generation of mature immunoregulatory dendritic cells.    Nat Immunol 2009; 10(1):48-57 doi 10.1038/ni.1674.-   18. Stanietsky N, Simic H, Arapovic J, Toporik A, Levy O, Novik A,    et al. The interaction of TIGIT with PVR and PVRL2 inhibits human NK    cell cytotoxicity. Proc Natl Acad Sci USA 2009; 106(42):17858-63 doi    10.1073/pnas.0903474106.-   19. Johnston R J, Comps-Agrar L, Hackney J, Yu X, Huseni M, Yang Y,    et al. The immunoreceptor TIGIT regulates antitumor and antiviral    CD8(+) T cell effector function. Cancer Cell 2014; 26(6):923-37 doi    10.1016/j.cce11.2014.10.018.-   20. Zhang B, Zhao W, Li H, Chen Y, Tian H, Li L, et al.    Immunoreceptor TIGIT inhibits the cytotoxicity of human    cytokine-induced killer cells by interacting with CD155. Cancer    Immunol Immunother 2016; 65(3):305-14 doi 10.1007/s00262-016-1799-4.-   21. Chan C J, Martinet L, Gilfillan S, Souza-Fonseca-Guimaraes F,    Chow M T, Town L, et al.

The receptors CD96 and CD226 oppose each other in the regulation ofnatural killer cell functions. Nat Immunol 2014; 15(5):431-8 doi10.1038/ni.2850.

-   22. Fuchs A, Cella M, Giurisato E, Shaw A S, Colonna M. Cutting    edge: CD96 (tactile) promotes NK cell-target cell adhesion by    interacting with the poliovirus receptor (CD155). J Immunol 2004;    172(7):3994-8.-   23. Machlenkin A, Uzana R, Frankenburg S, Eisenberg G, Eisenbach L,    Pitcovski J, et al. Capture of tumor cell membranes by trogocytosis    facilitates detection and isolation of tumor-specific functional    CTLs. Cancer Res 2008; 68(6):2006-13 doi    10.1158/0008-5472.CAN-07-3119.-   24. Ohtani H, Nakajima T, Akari H, Ishida T, Kimura A. Molecular    evolution of immunoglobulin superfamily genes in primates.    Immunogenetics 2011; 63(7):417-28 doi 10.1007/s00251-011-0519-7.-   25. Taube J M, Anders R A, Young G D, Xu H, Sharma R, McMiller T L,    et al. Colocalization of inflammatory response with B7-hl expression    in human melanocytic lesions supports an adaptive resistance    mechanism of immune escape. Sci Transl Med 2012; 4(127):127ra37 doi    10.1126/scitranslmed.3003689.-   26. Cerboni C, Fionda C, Soriani A, Zingoni A, Doria M, Cippitelli    M, et al. The DNA

Damage Response: A Common Pathway in the Regulation of NKG2D and DNAM-1Ligand Expression in Normal, Infected, and Cancer Cells. Front Immunol2014; 4:508 doi 10.3389/fimmu.2013.00508.

-   27. de Andrade L F, Smyth M J, Martinet L. DNAM-1 control of natural    killer cells functions through nectin and nectin-like proteins.    Immunol Cell Biol 2014; 92(3):237-44 doi 10.1038/icb.2013.95.-   28. Overwijk W W, Tsung A, Irvine K R, Parkhurst M R, Goletz T J,    Tsung K, et al. gp100/pmel 17 is a murine tumor rejection antigen:    induction of “self”-reactive, tumoricidal T cells using    high-affinity, altered peptide ligand. J Exp Med 1998;    188(2):277-86.

Example 6 Tumor Cell Killing Assay

The effect of an anti-human TIGIT antibody and CHA.7.518.1.H4(S241P),either alone or in combination, on tumor cell killing was assessed by anin vitro co-culture assay with human CMV-specific CD8⁺ T cells. TheHLA-A2⁺ target cell lines used in the assay were the melanoma cell line,Me1624, which stably expresses human PVR and PVRL2, and the pancreaticadenocarcinoma cell line, Panc05.04, which expresses endogenous levelsof human PVR and PVRL2. Both tumor cell lines were stably transducedwith a luciferase reporter gene through lentiviral transduction (SystemBiosciences). Me1624 and Panc05.04 cells were pulsed with the CMV pp65peptide at 0.0033 μg/ml or 0.01 μg/ml at 37° C. for 1 hour,respectively. Cells were then washed and plated at 20,000 cells/well. Abenchmark anti-human TIGIT antibody and CHA.7.518.1.H4(S241P) were addedto the culture in combination, or with a control hIgG4 isotype antibodyat 10 μg/ml. Human CMV-specific CD8⁺ T cells from three differentdonors, specified as Donor 4, Donor 72, and Donor 234 were added at100,000 cells/well. Co-cultures were incubated at 37° C. for 16 hours.After the incubation, plates were removed from the incubator and allowedto equilibrate to room temperature for 30 minutes. Bio-Glo luciferasesubstrate (Promega) was added to each well and the mixture equilibratedfor 10 minutes at room temperature protected from light. Luminesce orrelative light units (RLU) was quantified on an EnVision multi-labelreader (Perkin Elmer) with an ultra-sensitive luminescence detector.Percent specific killing was calculated by [(RLU for treatmentantibody—RLU for medium alone)/RLU for medium alone]×100.

Results

FIGS. 43A and B show the effect of the anti-TIGIT antibody andCHA.7.518.1.H4(S241P) treatment on killing of the Me1624 and Panc05.04cells, respectively. When added to the co-culture alone, both theanti-TIGIT antibody and CHA.7.518.1.H4(S241P) induced signficant T cellkilling of the tumor cell lines compared to the isotype controlantibody. For the anti-TIGIT antibody the percent specific killingranged from 19-41% for the Me1624 cells, and 3-44% for the Panc05.04cells across the 3 different CMV-reactive donors tested. ForCHA.7.518.1.H4(S241P), the percent specific killing ranged from 16-20%for the Me1624 cells, and 0.21-29% for the Panc05.04 cells. In somecases, an additive effect on tumor cell killing was observed in thecombined treatment of the anti-TIGIT antibody and CHA.7.518.1.H4(S241P).

To determine whether the effect of an anti-TIGIT antibody andCHA.7.518.1.H4(S241P) on tumor cell killing was dose-dependent, theassay was carried out with a 10 point, 2-fold dilution series for eachantibody starting at 0.5 μg/ml for the anti-TIGIT antibodies, and 10μg/ml for CHA.7.518.1.H4(S241P) (FIG. 44 ). Me1624 killing decreased ina dose-dependent manner when either anti-TIGIT antibody, BM26 orCPA.9.086, were combined with CHA.7.518.1.H4(S241P). More potent killingwas observed for the CPA.9.086 and CHA.7.518.1.H4(S241P) combinationwith an EC₅₀ of 0.40±0.49 nM, compared to the BM26 andCHA.7.518.1.H4(S241P) combination with an EC₅₀ of 2.6±1.7 nM.

Example 7 Biophysical Measurement of KD

KinExA equilibrium experiments were performed using a KinExA 3200instrument (Sapidyne Instruments, Boise, Id., USA) at 22° C. RecombinantHis-tagged human TIGIT was obtained from Sino Biologicals (Beijing,China) and reconstituted into 1×PBS. All antigen and antibody samplesfor KinExA analyses were prepared in degassed PBST buffer (PBS with0.05% tween 20) with 100 μg/mL filtered BSA and 0.02% sodium azide. Thesecondary detection antibody used was Alexa Flour 647-labeled goatanti-human IgG (H+L) (Jackson ImmunoResearch Laboratories) diluted 400-to 700-fold in the PBST buffer (with BSA and azide) described above froma 0.5 mg/mL stock in 1×PBS, pH 7.4. For each KinExA experiment, ˜20 μgof human TIGIT was diluted into 1 mL of 50 mM sodium carbonate, pH 9.2which was added directly to 50 mg of azlactone beads (Ultralink Support,Thermo Scientific, Rockford, Ill., USA) and rocked overnight at 4° C.After rocking, the beads were rinsed once with 1 M Tris buffer, pH 8.5,containing 10 mg/mL BSA and rocked for one hour at room temperature inthe same buffer. Coupled beads were added to the bead reservoir in theKinExA instrument and diluted to ˜30 mL with 1×HBS-N (0.01 M Hepes,0.15M NaCl, GE Healthcare) containing 0.02% sodium azide which was alsothe running buffer for the KinExA instrument. All antigen-coupled beadswere used immediately after preparation.

For two replicate measurements of K_(D) for CPA.9.086 (Table 1), 14concentrations of TIGIT ranging from 957 aM-212 pM were equilibrated atroom temperature for ˜72 hours with 2.5 pM CPA.9.086 binding sites and1.8 pM CPA.9.086 binding sites. For CPA.9.083, 14 concentrations ofTIGIT ranging from 478 aM-196 pM were equilibrated for ˜72 hours with1.8 pM CPA.9.083 binding sites. For duplicate measurements of thebenchmark antibody, BM26 hIgG4, 14 concentrations of TIGIT ranging from9.6 fM -3.53 nM were equilibrated for ˜72 hours with 20 pM BM26 bindingsites and 8.0 pM BM26 binding sites. For CHA.9.547.13, 14 concentrationsof TIGIT ranging from 10.5 fM-2.2 nM were equilibrated for ˜72 hourswith 8 pM mAb CHA.9.547.13 binding sites. The volume flowed through thebead pack for each equilibrated sample for all experiments ranged from 4mL to 11 mL at a flow rate of 0.25 mL/min. Data were fit with a 1:1“standard equilibrium” binding model using KinExA Pro software (Version4.2.10; Sapidyne Instruments) to estimate K_(D) and generate the 95%confidence interval (CI) of the curve fit.

Results

Both CPA.9.083 and CPA.9.086 bound to human TIGIT with femtomolarbinding affinity, while CHA.9.547.13 and BM26 bound with picmolaraffinity. Thus, CPA.9.083 and CPA.9.086 bound to human TIGIT with thehighest affinity of the four different antibodies tested.

TABLE 1 K_(D) measurements of anti-human TIGIT hIgG4 antibodiesdetermined by KinExA Antibody K_(D) ± 95% CI (n = 1) K_(D) ± 95% CI (n =2) CHA.9.547.13 18.8 ± 5.8 pM Not determined CPA.9.083 694 ± 277 fM Notdetermined CPA.9.086 553 ± 230 fM 665 ± 378 fM BM26 8.2 ± 2.8 pM 11.2 ±3.6 pM

EXAMPLE 8 Development and Functional Characterization of CPA.9.086, aNovel Therapeutic Antibody Targeting the Immune Checkpoint TIGIT

Background: TIGIT is a coinhibitory receptor that is highly expressed onlymphocytes, including effector and regulatory CD4+ T cells (Tregs),effector CD8+ T cells, and NK cells, that infiltrate different types oftumors. Engagement of TIGIT with its reported ligands, poliovirusreceptor (PVR) and PVR-like proteins (PVRL2 and PVRL3) directlysuppresses lymphocyte activation. PVR is also broadly expressed intumors, suggesting that the TIGIT-PVR signaling axis may be a dominantimmune escape mechanism for cancer. We report here the biophysical andfunctional characterization of CPA.9.086, a therapeutic antibodytargeting TIGIT. We also demonstrate that co-blockade of TIGIT and a newcheckpoint inhibitor, PVRIG, augments T cell responses.

Materials and Methods: Human phage display and mouse hybridoma antibodydiscovery campaigns were conducted to generate therapeutic anti-TIGITantibodies. The resulting antibodies were evaluated for their ability tobind to recombinant and cell surface-expressed human TIGIT with highaffinity. Cross-reactivity of the antibodies to cynomolgus macaque andmouse TIGIT was also examined. A subset of antibodies that bound withhigh affinity to human TIGIT, and cross-reactive to cynomolgus TIGITwere further characterized for their ability to block the interactionbetween TIGIT and PVR. Blocking antibodies were screened for theirability to enhance antigen-specific T cell activation in vitro eitheralone, or in combination with an anti-PVRIG antibody,CHA.7.518.1.H4(S241P).

Results: We identified a lead antibody, CPA.9.086, that binds to humanTIGIT with high femtomolar affinity. This antibody bound to TIGITendogenously expressed on human CD8+ T cells with higher affinity thantested benchmark antibodies, and was also cross-reactive to bothcynomolgus and mouse TIGIT. When tested for in vitro activity, CPA.9.086augmented cytokine secretion and tumor cell killing by CMV-specific CD8+T cells with superior or equivalent potency to the tested benchmarkantibodies. Combination of CPA.9.086 with an anti-PD1 antibody orCHA.7.518.1.H4(S241P) resulted in enhanced CMV-specific CD8+ T cellactivity. Furthermore, we demonstrated that TIGIT is predominantlyexpressed on Tregs and effector CD8+ T cells from solid tumors comparedto peripheral blood, suggesting that these populations will likely bepreferentially targeted by CPA.9.086.

Conclusion: We describe the development of a very high affinityantagonistic TIGIT antibody, CPA.9.086, that is currently in preclinicaldevelopment. We postulate that the femtomolar affinity of CPA.9.086could result in lower and less frequent dosing in patients. CPA.9.086can enhance human T cell activation either alone or in combination withother checkpoint antibodies. Thus, our data demonstrates the utility oftargeting TIGIT, PD1, and PVRIG for the treatment of cancer.

Example 9 Analysis of the TIGIT/PVRIG Axis in Human Cancers to SupportIndication Selection and Biomarkers for Co-Treatment

Background: PVRIG and TIGIT were identified by Compugen's PredictiveDiscovery Platform as immune inhibitory receptors and have been reportedto inhibit anti-tumor activity. We are pursuing clinical development ofantagonistic antibodies to PVRIG (e.g. CHA7.518.1.H4(S241P)) and toTIGIT (e.g. CPA.9.083.H4(S241P)). Here, we analyzed primary human cancertissues and immune cells to characterize expression in the TIGIT/PVRIGaxis to support indication selection and combination strategies forthese antibodies.

Methods: CHA7.518.1.H4(S241P) and CPA.9.083.H4(S241P) were identifiedbased on ability to block the interaction of PVRIG and TIGIT with theircognate ligands (PVRL2 and PVR respectively) and were screened for theirability to enhance antigen-specific CD8 T cell activation in aco-culture with tumor cell lines. Immunohistochemistry and Flowcytometry were performed to assess receptor/ligand expression indissociated bladder, breast, colorectal, head and neck, lung, kidney,ovarian, prostate, and stomach tumors.

Results: Among the cancers examined, PVRIG and PVRL2 expression washighest in endometrial, lung, kidney, ovarian, and head and neck cancerscompared to normal adjacent tissue. From dissociated tumors, PVRIGexpression was detected on T and NK TILs whereas PVRL2 expression wasdetected on CD45⁻ cells and myeloid cells. A co-expression analysis ofPVRIG, TIGIT, and PD1 demonstrated that PVRIG was co-expressed with bothTIGIT and PD1 and that PVRIG⁺TIGIT⁺PD1⁺ cells comprised a majorproportion of CD8 TILs. In comparison to PD-L1, PVRL2 expression wasmore prevalent across several cancer types and expression of PVRL2 wasdetected in PD-L1 negative samples. In vitro, combination ofCHA7.518.1.H4(S241P) with PD1 inhibitors or CPA.9.083.H4(S241P) enhancedCD8 cytokine production and cytotoxic activity, with the triplecombination of CHA7.518.1.H4(S241P), CPA.9.083.H4(S241P), and PD-1antibody yielding the greatest increase in functional activity. Severalimmune receptors were induced in response of PVRIG blockade byCHA7.518.1.H4(S241P) on CD8 T cells. Taken together, these data supportindication selection and combination strategies for CHA7.518.1.H4(S241P)and CPA.9.083.H4(S241P) and potential biomarkers that could beindicators of response.

Conclusions: In summary, we demonstrate that PVRIG and PVRL2 are inducedin the tumor microenvironment of human cancers, and the potential ofCHA7.518.1.H4(S241P) as a cancer therapeutic, either as a monotherapy oras a dual- or triple-combination therapy with antibodies targetingTIGIT, and PD-1. These data highlight the potential of this combinationapproach to expand the immune checkpoint inhibitor responsive cancerpatient population, including those who are non-responsive to PD-1inhibitors.

Example 10 PVRIG Expression is Associated with T Cell Exhaustion andSynergizes with TIGIT to Inhibit Anti-Tumor Responses

ABSTRACT

By employing a unique computational discovery platform, we identified anovel checkpoint receptor family comprised of 2 inhibitory receptors inthe nectin family, TIGIT and PVRIG. PVRIG and TIGIT are both expressedupon T cell activation, but display a difference in relative expressionamong T cell subsets and expression kinetics. PVRIG binds to PVRL2whereas TIGIT binds to several ligands, among which we observed that PVRis the dominant functional ligand for TIGIT. The distinct expressionprofile of PVRIG and a unique high affinity PVRIG-PVRL2 interactionsuggest that PVRIG has a unique role in regulating immunity. Using novelPVRIG−/− mice, we observed that genetic deficiency of PVRIG resulted inincreased T cell responses and reduced tumor growth in preclinicalmodels, demonstrating the potential of targeting this pathway in cancer.To further define a clinical niche for a PVRIG antagonist, weinterrogated the expression of TIGIT/PVRIG and PD-1 axis in human tumorsamples. Among the human cancers examined, PVRIG and TIGIT expression ontumor derived T cells were highest in endometrial, lung, kidney, andovarian cancers. A co-expression analysis of PVRIG, TIGIT, and PD1demonstrated that PVRIG was correlated and co-expressed with both TIGITand PD1 and that PVRIG⁺TIGIT⁺PD1⁺ cells comprised a major percentage ofCD8 tumor infiltrating lymphocytes (TILs). Interestingly, PVRIG and notTIGIT expression on CD8+ TILs were associated with an exhaustedEomes^(hi)T-bet^(lo) phenotype. PVR, PVRL2, and PD-L1 also displayedtissue specific differences in relative expression level, withendometrial and ovarian tumors having a higher ratio of PVRL2 expressionrelative to PVR or PD-L1. Culture of primary human TILs with anti-PVRIG(CHA7.518.1.H4(S241P)) and anti-TIGIT (CPA.9.083.H4(S241P)) antagonisticantibodies enhanced T cell function to a similar or greater magnitudecompared to PD-1 blockade. See, FIGS. 54 through 60 .

CHA7.518.1.H4(S241P) & CPA.9.083.H4(S241P) Target PVRIG and TIGIT in theNectin & Nectin-Like Family: Conclusions

PVRIG and TIGIT are non-redundant checkpoint receptors and promisingtargets for the treatment of cancer

In tumors with higher PVRL2 than PVR, the PVRIG/PVRL2 interaction couldbe more dominant and require direct targeting of PVRIG

Example 11 New Preclinical Data Demonstrating the Distinctive Featuresof the PVRIG Pathway in Immuno-Oncology and the Potential ofCHA7.518.1.H4(S241P) in Treating Multiple Solid Tumors

Data further strengthens rationale for Clinical Development Plan andBiomarker Strategy for CHA7.518.1.H4(S241P)

CHA7.518.1.H4(S241P) Shown to be Safe at High Doses in GLP ToxicityStudy

Provides new preclinical data demonstrating the distinctive features ofthe PVRIG pathway in Immuno-Oncology and the potential ofCHA7.518.1.H4(S241P), a first-in-class therapeutic antibody candidatetargeting PVRIG in treating multiple solid tumors. The data, presentedat the Keystone Symposia Conference, A3: T Cell Dysfunction, Cancer andInfection, being held Jan. 16-20, 2018 (an provided in Example 9 andrelated figures), demonstrate the possible dominance of the PVRIG/TIGITaxis in immuno-oncology and support the Company's clinical developmentprogram and biomarker strategy for CHA7.518.1.H4(S241P) as a monotherapyand in combination with CPA.9.083.H4(S241P).

The poster titled “PVRIG Expression is Associated with T Cell Exhaustionand Synergizes with TIGIT to Inhibit Anti-Tumor Responses” (Poster no.2028) includes data showing that expression of PVRL2, the ligand forPVRIG, is more dominant in several tumor types including lung, breast,endometrial, and ovarian, than the expression of PVR, the ligand forTIGIT. These results suggest that PVRIG may be the dominant checkpointin patient populations with tumor expressing elevated PVRL2, many ofwhich are not responsive to PD-1 inhibitors. Therefore, these patientsmay have an increased likelihood of responding to CHA7.518.1.H4(S241P)as a monotherapy treatment.

In addition, expression studies show that PVRIG and TIGIT and theirrespective ligands are commonly expressed in the tumor types listedabove as well as kidney and head & neck cancers, indicating that inpatient populations where the two pathways are operative the blockade ofboth TIGIT and PVRIG may be needed in order to sufficiently stimulatethe anti-tumor immune response. Furthermore, data also indicates thatexhausted TILs found in multiple tumor types are largely co-expressingthe triple three checkpoints -, TIGIT, PD-1 and PVRIG, furthersupporting the relevance of a triple combination in such patientpopulations.

Our growing understanding of the PVRIG-TIGIT axis and the interplaybetween the various axis components also shed light on the evolution ofthe PVRIG/PVRL2 pathway in the transition from mouse to humans,resulting in a less active pathway in mice. Our data clearlydemonstrates that the mouse biology in connection of this pathway isunderestimating the human impact this pathway may have on anti-tumorimmunity, suggesting that CHA7.518.1.H4(S241P) may have an even greatertherapeutic impact than the one seen in preclinical studies.

“The potential dominance of the PVRIG pathway and its interactions withthe TIGIT and PD-1 pathways demonstrated in our preclinical studiescoupled with the expression profiles provide the biological rationale tosupport our clinical approach to test CHA7.518.1.H4(S241P) as amonotherapy and in dual and triple combination as we are preparing toinitiate our clinical Phase lb trial. While keeping our all cornerstrial design, our biomarker strategy will be driven by these expressionprofiles, in order to enrich for patients most likely to respond toCHA7.518.1.H4(S241P),” stated Anat Cohen-Dayag, PhD, President and CEOof Compugen. “We are also encouraged by results of the GLP toxicitystudy for CHA7.518.1.H4(S241P), showing it to be safe at high doses. Ourdata lead us to believe that the PVRIG pathway and CHA7.518.1.H4(S241P),our first-in-class therapeutic antibody, may hold significant clinicalvalue as the basis of new cancer immunotherapies to meet the needs ofpatient populations non-responsive or refractory to current immunecheckpoint inhibitor therapy.”

About CHA7.518.1.H4(S241P) and CPA.9.083.H4(S241P)

CHA7.518.1.H4(S241P) is a humanized hybridoma antibody that binds withhigh affinity to PVRIG, a novel B7/CD28-like immune checkpoint targetcandidate discovered by Compugen, indicating blockage of this target'sinteraction with PVRL2. Blockade of PVRIG by CHA7.518.1.H4(S241P) hasdemonstrated potent, reproducible enhancement of T cell activation,consistent with the desired mechanism of action of activating T cells inthe tumor microenvironment to generate anti-tumor immune responses. Inaddition, CHA7.518.1.H4(S241P) combined with antagonist anti-PD-1antibodies has demonstrated synergistic effect on human T cellstimulation, indicating the potential of these combinations to furtherenhance immune response against tumors.

CPA.9.083.H4(S241P), Compugen's antibody targeting TIGIT, was developedfor combination use with CHA7.518.1.H4(S241P). Preclinical data stronglysupport the dual blockade of the two negative costimulatory arms of theaxis—TIGIT and PVRIG—that results in a more robust T cell response toantigen stimulation, and therefore may result in an enhanced anti-tumorimmune response.

Example 12 In Vivo Efficacy and Survival with Triple Combo TreatmentRationale and Objectives

This example provides regarding whether the combination of mouse TIGIT,PVRIG and PD-L1 blockade can significantly enhance tumor growthinhibition (TGI) and survival in a syngeneic mouse tumor model.

Protocols Animals

Female mice of 5 week-age were purchased from Charles RiverLaboratories. The mice were housed at the Compugen USA animal facilitywith food and water provided, ad libitum, and acclimated for a minimumof 6 days prior to initiation of the study. All studies were approved bythe Institutional Animal Care and Use Committee at the Compugen USA(South San Francisco, Calif.).

Syngeneic Mouse Tumor Model

5×10⁵ CT26 colon carcinoma (ATCC) cells were inoculated subcutaneously(s.c.) into the right flank of female Balb/c mice, and grown for up to 8days. Mice with tumors measuring 30-60 mm³ were randomized (day ofrandomization designated day 0) into 3 groups of 10 mice per group.Animals received a 200 μL intraperitoneal (i.p.) injection of either amouse IgG1 (mIgG1) isotype control antibody (referred to as Synagis), adual combination of an anti-TIGIT mIgG1 and anti-PVRIG mIgG1 antibodies,or a triple combination of anti-TIGIT mIgG1, anti-PVRIG mIgG1, andanti-PD-L1 mIgG1 antibodies. The anti-TIGIT mIgG1 antibody is a chimericversion of CPA.9.086 that contains the human variable heavy and lightchains of CPA.9.086 and the constant region of mIgG1. The antibodieswere administered at fixed-doses of 10 mg/kg anti-TIGIT, 10mg/kganti-PVRIG, and a suboptimal dose of anti-PD-L1 at 3 mg/kg starting onday 8 three times per week for 2 weeks. Tumor growth is determined bycaliper measurement of length (L) and width (W); wherein the tumor sizeis calculated with the formula (L×W²)/2. Tumor size were not allowed toexceed 2000 mm³, which was designated as the study endpoint, and micewere subsequently euthanized.

Statistical Analysis

Two-way ANOVA with repeated measures, followed by two-way ANOVA withrepeated measures for selected pairs of groups was performed using prismsoftware. Analyses of tumor growth measurements were performed bycomparing tumor volumes measured on the last day on which all studyanimals were alive. Statistical differences in percentage of mice tumorfree were determined by a Log Rank Mantel—Cox test.

Results and Summary

In this study, the effect of blocking three different immune checkpointpathways, TIGIT, PVRIG and PD-L1, on TGI and survival in mice wasexamined. Using the mouse colon carcinoma CT26 model, the combination ofthe anti-TIGIT mIgG1 with the anti-PVRIG mIgG1 resulted in small butsignificant TGI (20.7% TGI at day 25) compared to the mice dosed withthe isotype control antibody. When the three antibodies were combined,TGI increased to 58.3% at day 25, which was statistically andsignificantly efficacious compared to the dual combination (p<0.001 bytwo-way ANOVA at day 25). Although none of the mice were tumor free (CR)by the end of the study (day 28), the enhanced efficacy in triplecombination was associated with increased survival compared to dualcombination. Triple blockade demonstrated significant enhancement ofoverall survival with 90% survival at the study endpoint (day 28). Inaddition to the antitumor efficacy reported here, there was nosignificant body weight changes in all groups observed (data not shown).Taken together, the triple combination was well tolerated and produced asuperior antitumor effect in colon carcinoma in vivo compared to thedual combination of TIGIT and PVRIG blockade alone. See, FIG. 61 .

1.-58. (canceled)
 59. A method of treating cancer in a patientcomprising: a) providing a biopsy from said patient comprising tumorcells; b) measuring the frequency of PD-L1 positive tumor cells orimmune cells in said biopsy; c) if said frequency of PD-L1 positivetumor cells or immune cells is greater than 1% compared to staining thesame tumor cells with a relevant isotype control antibody for theantibodies used, administering a triple combination therapy comprisingan anti-TIGIT antibody, an anti-PVRIG antibody, and an anti-PD-1antibody; and d) if said frequency of PD-L1 positive tumor cells orimmune cells is less than 1% compared to staining the same tumor cellswith a relevant isotype control antibody for the antibodies used,administering a double combination therapy comprising an anti-TIGITantibody and an anti-PVRIG antibody.
 60. A method of treatment forcancer in a patient comprising administering the triple combinationcomprising an anti-TIGIT antibody, an anti-PVRIG antibody and ananti-PD-1 antibody, wherein said anti-TIGIT antibody is an antibodychosen from at least one of CPA.9.083.H4(S241P), CPA.9.086.H4(S241P),CHA.9.547.7.H4(S241P) and CHA.9.547.13.H4(S241P).
 61. The method oftreatment according to claim 60, wherein said anti-TIGIT antibody isCPA.9.086.H4(S241P).
 62. The method of treatment according to claim 60,wherein said anti-TIGIT antibody is CPA.9.083.H4(S241P).
 63. The methodof treatment according to claim 60, wherein said anti-PVRIG antibody isan antibody chosen from at least one of CHA.7.518.1.H4(S241P) andCHA.7.538.1.2.H4(S241P).
 64. The method of treatment according to claim60, wherein said anti-PD-1 antibody is an antibody chosen from at leastone of pembrolizumab and nivolumab.
 65. The method of treatmentaccording to claim 60, wherein said antibodies are administeredsimultaneously.
 66. The method of treatment according to claim 60,wherein said antibodies are administered as separate infusions or as oneinfusion of a mixture of the antibodies.
 67. The method of treatmentaccording to claim 60, wherein said antibodies are administeredsequentially.
 68. The method of treatment according to claim 67, whereinthe antibodies are administered sequentially over a period of hours ordays.
 69. The method of treatment according to claim 60, wherein saidcancer is selected from the group consisting of prostate cancer, livercancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer,breast cancer, triple negative breast cancer, pancreatic cancer, stomach(gastric) cancer, cervical cancer, head and neck cancer, thyroid cancer,testis cancer, urothelial cancer, lung cancer (small cell lung,non-small cell lung), melanoma, non melanoma skin cancer (squamous andbasal cell carcinoma), glioma, renal cancer (RCC), lymphoma (NHL or HL),Acute myeloid leukemia (AML), T cell Acute Lymphoblastic Leukemia(T-ALL), Diffuse Large B cell lymphoma, testicular germ cell tumors,mesothelioma, esophageal cancer, Merkel Cells cancer, MSI-high cancer,KRAS mutant tumors, adult T-cell leukemia/lymphoma, and Myelodysplasticsyndromes (MDS).
 70. The method of treatment according to claim 60,wherein said cancer is selected from the group consisting of ovariancancer, triple negative breast cancer, stomach (gastric) cancer, lungcancer (small cell lung, non-small cell lung), Merkel Cells cancer,MSI-high cancer, KRAS mutant tumors, adult T-cell leukemia/lymphoma, andMyelodysplastic syndromes (MDS).
 71. The method of treatment accordingto claim 60, wherein the antibodies are provided in an administrationkit with dosage units of each antibody, either packaged separately inindividual dosage units, or together, as a mixture of antibodies as asingle dosage unit.